Anthelmintic compositions and methods

ABSTRACT

Compositions and methods for treating or reducing the severity or likelihood of occurrence of a parasitic worm or helminth infection in a subject are described. The methods include administering to the subject a therapeutically effective amount of a killed or inactivated recombinant bacterium expressing a crystal protein such as a  Bacillus thuringiensis  crystal protein (Cry) in the cytosol of the bacterium. The crystal proteins may be full length, truncated, variant, or sub-variant Cry proteins. Examples of crystal proteins include Cry5B, Cry21, Cry14A, Cry6A, and Cry13A. The recombinant bacteria may be treated with an anti-microbial agent before or during administration to a subject.

RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 filing of International Patent Application No. PCT/US2017/013436, filed Jan. 13, 2017, which claims the benefit of priority to U.S. Provisional Patent Application No. 62/279,597, filed Jan. 15, 2016. The entire content of these applications are incorporated herein by reference for all purposes.

BACKGROUND

Soil-transmitted helminthes (STHs) that parasitize the GI tract of humans infect 2.3 billion of the poorest peoples and >400,000,000 of the poorest children worldwide. (Hall, A., et al. Matern Child Nutr 4 Suppl 1, 118-236 (2008)) Infected children can exhibit growth stunting, retarded cognitive development, lethargy, malnutrition, increased school absenteeism, and vulnerability to secondary infections. (Bethony, J. et al. Lancet 367, 1521-32 (2006); Hotez, P. J. Forgotten people, Forgotten diseases. (2008)) Pregnant women who are infected are at increased risk for low birth-weight babies and for maternal and infant mortality. (Brooker et al., PLoS Negl Trop Dis 2, e291 (2008)). Infected individuals have lower energy, lower productivity, and immune defects that result in increased virulence of HIV/AIDS and a higher likelihood of contracting malaria and tuberculosis (Stothard et al., Ann Trop Med Parasitol 103, 357-60 (2009); Moran, M. et al., G-finder Report (2009)); STHs thus trap large populations of the developing world in poverty. The common link of STH transmission is poor sanitation, which requires a massive investment in infrastructure and public health.

Conventional chemotherapy approved by the World Health Organization for STH infections in humans involves treatment with benzimidazoles (e.g., albendazole, mebendazole) or nicontinic acetylcholine receptor (nAChR) agonists (pyrantel, levamisole). (Keiser and Utzinger, JAMA 299, 1937-48 (2008)). These compounds, however, lack full efficacy against most human STH parasites. Reports in humans of resistance to both classes of drugs are increasing (e.g., Tanzania, 2010 (Stothard et al., Ann Trop Med Parasitol 103, 357-60 (2009)), potentially rendering ineffective current strategies for controlling STH infections. A notable challenge in this field is that the infected populations are among the poorest in the world, and economic incentives to develop new drugs are low (˜$700,000/year is spent to develop new drugs against human STHs (Moran, M. et al. G-finder Report (2009)). The poverty of infected populations demands that STH therapeutics be safe, effective, and also inexpensive; highly stable even in the absence of a cold chain; transportable through distribution routes to infected populations; and amenable to culturally acceptable delivery systems.

Crystal (Cry) proteins made by the soil bacterium Bacillus thuringiensis (Bt) may be candidate agents that provide safe and effective treatment of STHs. Cry proteins have been in use for 60+ years as safe, natural, organic insecticides for control of crop pests, mosquitoes, and black flies. (Roh, J. Y., et al. J MICROBIOL BIOTECHNOL 17, 547-59 (2007)). They are also effective against nematodes. (Wei, J. Z. et al. PROC NATL ACAD SCI 100, 2760-5 (2003)). Cry proteins are non-toxic to vertebrates and are EPA approved for expression in transgenic food (e.g., corn, potato). (Mohamadzadeh et al. PNAS 106, 4331-6 (2009); Betz F. S., et al. REGUL TOXICOL PHARMACOL 32, 156-73 (2000)). They are stable and cheap to mass-produce. Activity of Cry proteins against nematode plant parasites and against helminthes has been described, e.g., in WO2007/062064; US2010/0024075; WO2010/053517; and US2011/0263489; see also, e.g., Li, X.-Q. et al., 2008 Biol. Control 47:97-102, which describes activity of a Cry5B protein truncated at amino acid residue 698 against C. elegans and plant parasitic nematodes.

Two Cry proteins, Cry5B and Cry21A, are highly potent anthelmintics in vivo. (See Cappello, M. et al. P ROC N ATL A CAD S CI USA 103, 15154-9 (2006); Hu, Y., et al. PLOS NEGL TROP DIS 4, e614 (2010); and Hu, Y., et al. P ROC N ATL A CAD S CI USA 107, 5955-60 (2010)). Cry5B is effective against three intestinal nematodes, Ancylostoma ceylanicum hookworms in hamsters, Heligmosomoides bakeri in mice, and Ascaris suum parasites in pigs, and is 3×-60,0000× more potent than known chemical anthelmintics in a single dose. (See Cappello, M. et al. P ROC N ATL A CAD S CI 103, 15154-9 (2006); Hu, Y., et al. PL O S N EGL T ROP D IS 4, e614 (2010); Hu, Y., et al. PL O S N EGL T ROP D IS 6(11), e1900 (2012); and Urban, J., et al PL O S N EGL T ROP D IS 7(6), e2263 (2013)). Importantly, screens for Cry-resistance mutations in the nematode Caenorhabditis elegans indicate that nematodes are 3-20× less likely to develop resistance to Cry proteins than to benzimidazoles or nAChR agoinsts. (Hu, Y., et al. P ROC N ATL A CAD S CI 107, 5955-60 (2010)). Furthermore, Cry5B is able to overcome benzimidazoles and nAChR agonist resistance in nematodes (Hu, Y., et al. P ROC N ATL A CAD S CI 107, 5955-60 (2010)).

Despite the established anthelmintic biological activity of Cry proteins, significant challenges remain with respect to effective delivery of intact, biologically active Cry proteins into the gastrointestinal (GI) tract for treating STHs. These proteins typically have molecular weights of ˜135 kDa in their protoxin (unprocessed) forms and ˜70 kDa in their active (processed) forms, creating technical difficulties for delivery to the GI lumen via known routes of administration, including problems arising from degradation, poor absorption, clearance mechanisms and other impediments. Moreover, the cost and scalability of Cry protein expression and purification limits its application as a practical STH therapy in the developing world where treatments must be available at a very low costs (less than $1/dose) and in very large quantities to treat a large and poor patient population.

A cheap, simple, and scalable way to deliver Cry proteins is to express it in B. thuringiensis, which is ideally suited to express very high levels of Cry protein and which is already fermented cheaply on a massive scale for environmental release. However, B. thuringiensisis is very closely related to the human pathogen Bacillus cereus and contains many of the enterotoxin genes that causes food poisoning in humans Feeding people large quantities of fully active B. thuringiensis therefore has pathogenic potential. Accordingly, there remains an urgent need in the art for for new approaches to delivering protein therapeutics such as anthelmintic proteins to the GI tract.

SUMMARY

The instant disclosure improves upon the art by providing antihelminthic compositions that are both safe and effective for oral delivery. In particular, the instant disclosure is based on the surprising discovery that a non-sporulating bacterium expressing a nematicidal protein (e.g., a heterologous B. thuringiensis crystal protein) can be inactivated or killed to reduce or eliminate its toxicity when orally administered to a human subject but without altering the anti-helminthic activity of the nematicidal protein. Notably, the anti-nematicidal efficacy of the killed bacterial product against a particular parasitic worm or helminth is superior to that of purified nematicidal protein.

In exemplary embodiments, the inactive or killed bacterium is genetically engineered such that nematicidal protein is made and trapped in the cytosol of the bacterium. The protein may be retained in the cytosol or released, e.g., if the bacterium is broken open following digestion. The non-sporulating bacterium can be a sporulation-defective variant of a Gram positive bacterium, such as a Bacillus sp., including B. thuringiensis. For example, the bacterium can be a sporulation mutant, such as a spo0A- mutant. Alternatively, non-sporulating baceterium can be a Gram-negative bacterium (e.g., E.coli or P. fluorescens).

In certain aspects, the instant disclosure provides an orally-available pharmaceutical composition comprising a killed or inactivated and non-sporulating bacterium that is genetically engineered to express a nematicidal protein. In certain embodiments, the killed or inactivated bacterium is genetically engineered to have a genetic mutation that results in a defect in sporulation such that the nematicidal protein is expressed and trapped in the cytosol of the bacterium. In one embodiment, the nematicidal protein is under control of a promoter that is actively and/or highly expressed prior to the sporulation phase of a bacterium, e.g., during the vegetative growth or stationary phase. In certain embodiments, the promoter is heterologous (i.e., a non-sporulation specific promoter). In one embodiment, the promoter is a Cry3A, GerA, GNAT, or TadA promoter. In one embodiment, the nematicidal protein is a cytoplasmic Cry crystal protein. In one embodiment, the cytoplasmic Cry protein is selected from the group consisting of Cry5B, Cry5C, Cry5D, Cry6A, Cry13A, Cry14A, Cry21A, Cry21B, and Cry55A. In one embodiment, the inactivated bacterium is Bacillus sp. In one embodiment, the inactivated bacterium is Bacillus thuringiensis (Bt).

In one embodiment, the genetic mutation resulting in a defect of sporulation is the deletion or inactivation of one or more genes selected from the group consisting of: kinA, kinB, spo0A, spo0B, spo0E, spo0F, spo0J, spo0M spoIIB, spoIID, spoIIE, spoIIF, spoIIG, spoIIL, spoIIM, spoIIIA, spoIIIB, spoIIIE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK. In one embodiment, the genetic mutation resulting in a defect of sporulation is the deletion or inactivation of the spo0A gene. In one embodiment, the composition is encapsulated by a pharmaceutical grade capsule in a dry powdered form.

In another aspect, a method for producing a pharmaceutical composition, the method comprising: exposing a bacterium to an antimicrobial agent, wherein the bacterium is genetically engineered to express a nematicidal protein. In certain embodiments the inactivated bacterium is genetically engineered to have a genetic mutation that results in a defect in sporulation. In another embodiment, the method further comprises lyophilizing the bacterium. In another embodiment, the method further comprises encapsulating the bacterium in a pharmaceutical-grade capsule. In one embodiment, wherein the antimicrobial agent is selected from the group consisting of: an antimicrobial compound; and gamma irradiation. In one embodiment, the antimicrobial compound is a food-grade antibiotic. In another embodiment, the antimicrobial compound is a beta-lactam antibiotic. In another embodiment, the antimicrobial compound is a terpene. In another embodiment, the terpene is selected from the group consisting of thymol, eugenol, geraniol, carvacrol, and citral, or a combination thereof. In another embodiment, the terpene is carvacrol. In another embodiment, the nematicidal protein is under control of a promoter that is actively and/or highly expressed prior to the sporulation phase of a bacterium, e.g., during the stationary phase. In certain embodiments, the promoter is heterologous. In one embodiment, the promoter is a Cry3A, GerA, GNAT, or TadA promoter.

In one embodiment, the nematicidal protein is a cytoplasmic crystal protein. In another embodiment, the cytoplasmic crystal protein is selected from the group consisting of Cry5B, Cry5C, Cry5D, Cry6A, Cry13A, Cry14A, Cry21A, Cry21B, and Cry55A. In another embodiment, wherein the inactivated bacterium is Bacillus sp. In another embodiment, the inactivated bacterium is Bacillus thuringiensis (Bt). In another embodiment, the genetic mutation resulting in a defect of sporulation is the deletion or inactivation of one or more genes selected from the group consisting of: kinA, kinB, spo0A, spo0B, spo0E, spo0F, spo0J, spo0M spoIIB, spoIID, spoIIE, spoIIF, spoIIG, spoIIL, spoIIM, spoIIIA, spoIIIB, spoIIIE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK. In another embodiment, the genetic mutation resulting in a defect of sporulation is the deletion or inactivation of the spo0A gene.

In another aspect, the disclosure provides a method of treating a parasitic worm infection in a subject comprising: administering to the subject a therapeutically effective amount of a composition comprising a killed or inactivated bacterium that is genetically engineered to express a nematicidal protein. In certain embodiments, the inactivated bacterium is genetically engineered to have a genetic mutation that results in a defect in sporulation. In one embodiment, the nematicidal protein is under control of a promoter that is actively and/or highly expressed prior to the sporulation phase of a bacterium, e.g., during the stationary phase. In certain embodiments, the promoter is heterologous. In one embodiment, the promoter is a Cry3A, GerA, GNAT, or TadA promoter.

In another embodiment, the nematicidal protein is a cytoplasmic crystal protein. In another embodiment, wherein the cytoplasmic crystal protein is selected from the group consisting of Cry5B, Cry5C, Cry5D, Cry6A, Cry13A, Cry14A, Cry21A, Cry21B, and Cry55A. In another embodiment, the inactivated bacterium is Bacillus sp. In another embodiment, the inactivated bacterium is Bacillus thuringiensis (Bt). In another embodiment, the genetic mutation resulting in a defect of sporulation is the deletion or inactivation of one or more genes selected from the group consisting of: kinA, kinB, spo0A, spo0B, spo0E, spo0F, spo0J, spo0M spoIIB, spoIID, spoIIE, spoIIF, spoIIG, spoIIL, spoIIM, spoIIIA, spoIIIB, spoIIIE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK. In another embodiment, the genetic mutation resulting in a defect of sporulation is the deletion or inactivation of the spo0A gene. In another embodiment, the composition is encapsulated by a pharmaceutical grade capsule.

The parasitic worm or helminth infection can be an infection caused by a parasitic worm or helminth selected from the group consisting of roundworm, whipworm, hookworm, Ascaris, pinworm, Strongyloides, Schistosome, and trematodes. The parasitic worm or helminth can be selected from the group consisting of hookworm Ancylostoma duodenale, hookworm Ancylostoma ceylanicum, hookworm Necator americanus, whipworm, Trichuris trichiura, roundworm Ascaris lumbricoides, threadworm Strongyloides stercoralis, and pinworm Enterobius vermiculari.

In the method, the subject can be a mammal, such as a feline, rodent, canine, bovine, equine, swine, caprine, ovine, and primate; for example, the mammal can be a human.

The crystal protein can be selected from the group consisting of Cry5B, Cry5C, Cry5D, Cry6A, Cry13A, Cry14A, Cry21A, Cry21B, and Cry55A. The crystal protein can be truncated; it can also be a variant. When truncated, the crystal protein can be truncated after a conserved amino acid sequence of block 5. The truncated crystal protein can be missing the last 10 amino acids of the C-terminus; in some cases the truncated crystal protein is truncated between the end of conserved block 5 and the C-terminus of the full length protein. When referring to block 5, the conserved amino acid sequence can be DRIEF (SEQ ID NO:23) or DRLEF (SEQ ID NO:24). The truncated crystal protein can have toxic activity that is at least 10% or more of the toxic activity of a corresponding full-length protein. The truncated crystal protein can be truncated at the N-terminus, such as when the truncated crystal protein does not contain the first 5 amino acids of the N-terminus. Such a truncated crystal protein can be truncated at the C-terminus. The crystal protein can be selected from the group consisting of:

a. Cry5B and wherein the Cry5B includes at least amino acids 30 through about 693 of SEQ ID NO:1

b. Cry6A and wherein the Cry6A comprises the amino acid sequence set forth in SEQ ID NO:2 or includes at least amino acids 30 through about 395, 415 or 435 of SEQ ID NO:6,

c. Cry13A and wherein the Cry13A includes at least amino acids 30 through about 688 of SEQ ID NO:2,

d. Cry14A and wherein the Cry14A includes at least amino acids 30 through about 675 of SEQ ID NO:3,

e. Cry21A and wherein the Cry21A includes at least amino acids 30 through about 685 of SEQ ID NO:4, and

f. Cry21A and wherein the Cry21A includes at least amino acids 30 through about 685 of SEQ ID NO:5.

In the disclosed method, the bacterium can be treated with the anti-microbial agent prior to administration of the bacterium to the subject. In some embodiments, the bacterium and the anti-microbial agent are co-administered to the subject. The anti-nematicidal activity of the nematicidal protein is preferably unaffected by the anti-microbial agent. As used herein, an “anti-microbial agent” may be a chemical or physical agent. An example of a chemical anti-microbial agent is a bacteriocidal agent, such as a beta-lactam antibiotic. An example of a physical anti-microbial agent is irradiation (e.g., gamma or U.V. irradiation) or heat treatment. In some embodiments, the anti-microbial agent is iodine or a terpene or formaldehyde. In the case of iodine, it can be Lugol's iodine. In the case of terpene, it can be one selected from the group consisting of thymol, eugenol, geraniol, carvacrol, and citral, or a combination thereof. In some embodiments, the terpene is carvacrol. In certain embodiments, the anti-microbrial agent is a food-grade antibiotic. In the disclosed method, the recombinant bacterium is killed by the anti-microbial treatment.

The disclosed method can further comprise administering an additional therapeutic agent, such as an agent selected from the group consisting of a bacterium expressing, or capable of expressing, a crystal protein, a small molecule, and a polypeptide. An example of such a therapeutic agent is a nicotinic acetylcholine receptor agonist, such as a member of the levamisole family of nicotinic acetylcholine receptor agonists; an example would be levamisole. In other embodiments, the nicotinic acetylcholine receptor agonist is pyrantel or tribendimidine.

In another aspect, disclosed herein is a method of treating hookworm infection in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-microbial agent treated recombinant B. thuringiensis spo0A- bacterium that is engineered to express a Cry5B crystal protein.

In another aspect, disclosed herein is a method of reducing the severity of a parasitic worm or helminth infection in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-microbial agent treated recombinant bacterium that is engineered to express a crystal protein.

In another aspect, disclosed herein is a method of preventing a parasitic worm or helminth infection in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-microbial agent treated recombinant bacterium that is engineered to express a crystal protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B FIG. 1A depicts the positions of conserved blocks among certain Cry proteins. de Maagd, R. A., et al. “How Bacillus thuringiensis has evolved specific toxins to colonize the insect world.” Trends in Genetics 17(4): 193-99, 195 (FIG. 2a ) (April 2001). FIG. 1B illustrates the positions of conserved blocks among certain Cry proteins. Schnepf, E., et al. “Bacillus thuringiensis and Its Pesticidal Crystal Proteins.” Microbiology and Molecular Biology Reviews 62(3): 775-806, 781 (FIG. 3) (September 1998).

FIG. 2 illustrates the amino acid sequence of Cry5Ba1 [SEQ ID NO:1].

FIG. 3 illustrates the amino acid sequence of Cry13Aa1 [SEQ ID NO:2].

FIG. 4 illustrates the amino acid sequence of Cry14Aa1 [SEQ ID NO:3].

FIGS. 5A-C FIG. 5A illustrates the amino acid sequence of Cry21Aa1 [SEQ ID NO:4]. FIG. 5B illustrates the amino acid sequence of Cry21Aa2 (98% identical to Cry21Aa1) [SEQ ID NO:5]. FIG. 5C illustrates the amino acid sequence of Cry6A [SEQ ID NO:6].

FIG. 6 depicts PY79-Cry5B bioactivity in vitro against C. elegans. The results shown are from dose-dependent mortality assays plotting % live C. elegans (y axis) versus Cry5B concentration (x axis). The PY79-vector strain (vector-PY79) lacked Cry5B (0 μg/ml). Each data point represents the average for three independent experiments with ˜75 to 90 C. elegans organisms per experiment (˜225 to 270 organisms per data point). Error bars represent standard errors.

FIGS. 7A-C shows that PY79-Cry5B had a dose-dependent therapeutic effect against hookworm infection in hamsters. FIG. 7A depicts intestinal hookworm burdens in nine hamsters following treatment with PY79-vector or PY79-Cry5B (10 mg/kg Cry5B) (error bars in all panels show standard errors). The average worm burdens were 18.6±2.6 and 1.3±0.3 for PY79-vector and PY79-Cry5B, respectively. FIG. 7B depicts fecal egg counts on day −1, day +1, and day +3 relative to the day of treatment. The actual egg counts for PY79-vector and PY79-Cry5B were 965±193 and 1,044±99, respectively, on day −1, 1,055±230 and 94±60, respectively, on day +1, and 1,055±227 and 100+42, respectively, on day +3. EPG, eggs per gram of feces. FIG. 7C depicts in vivo dose-response experiment with 12 hamsters. The average worm burdens for PY79-vector and PY79-Cry5B at Cry5B concentrations of 0.4 mg/kg, 1.4 mg/kg, and 4 mg/kg were 27.0±3.2, 15.7±7.0, 8.3±0.9, and 5.7±0.9, respectively.

FIG. 8 shows results from an in vivo bioassay of intestinal worm burdens in golden Syrian hamsters infected with Ancylostoma ceylanicum hookworms as described by Hu et al. (2012 PLoS Negl. Trop. Dis. 6:e1900. doi:10.137/journal.pntd.0001900). The four groups (in black, n=7 per group) shown are the intestinal worm burdens from the groups of infected hamsters treated with purified full-length B. thuringiensis Cry5B protein (prepared according to Griffitts et al., 2001 Science 293:860; for sequence see FIG. 2) at a single dose of 1 mg (solid circles, 10 mg/kg), 3 mg (solid upright triangles, 30 mg/kg), or 10 mg (solid inverted triangles, 100 mg/kg) (715 nmoles/kg), or with placebo (solid squares, ddH₂O), respectively. The treatments were conducted on day 16 P.I. and intestinal worm burdens assessed on day 21 P.I. The worm burdens in each hamster are indicated with a separate symbol. Long horizontal bars represent mean worm burdens; smaller bars indicate SEM (standard error of the mean).

FIG. 9 shows dose-response results for indicated dosages of unfractionated Cry5B-containing spore-crystal lysates (SCL) in the in vivo bioassay of intestinal worm burdens in golden Syrian hamsters infected with Ancylostoma ceylanicum hookworms. The assay was performed according to Hu et al. (2012 PLoS Negl. Trop. Dis. 6:e1900. doi:10.137/journal.pntd.0001900) except instead of purified Cry5B protein the animals received the indicated dosages, via gavage, of Cry5B spore-crystal lysates obtained from cultured Bacillus thuringiensis cells that were transformed with a low copy plasmid that expressed B. thuringiensis Cry5B and then grown to sporulation phase, at which point the cells lysed releasing spores, crystals, and bacterial lysate (spore crystal lysate, SCL). The amounts of Cry5B gavaged were determined by taking known volumes of spore crystal lysates, resolving full length Cry5B protein by SDS PAGE, and quantitating the amount of protein in the Cry5B band relative to known amounts of bovine serum albumin (BSA) standards on the gel.

FIG. 10 shows results from the in vivo bioassay of intestinal worm burdens in golden Syrian hamsters infected with Ancylostoma ceylanicum hookworms (Hu et al., 2012 PLoS Negl. Trop. Dis. 6:e1900. doi:10.137/journal.pntd.0001900) following treatment with two different dosages of Cry5B spore-crystal lysates obtained from cultured Bacillus thuringiensis natto cells that were transformed with a low copy plasmid that expressed B. thuringiensis Cry5B and then grown to sporulation phase.

FIG. 11 shows data obtained in vitro using the C. elegans mortality assay described in FIG. 6 to evaluate the effects on C. elegans of purified Cry5B protein (prepared according to Griffitts et al., 2001 Science 293:860; for sequence see FIG. 2) when combined in a mixture either with sporulated B. thuringiensis HD1 or with sporulated B. subtilis PY79. For each data point, the number of spores (HD1 or PY79) was held constant and the quantity of Cry5B was titrated (x-axis).

FIG. 12 shows the effects of Cry14A on an in vivo bioassay of intestinal worm burdens in golden Syrian hamsters infected with Ancylostoma ceylanicum hookworms.

FIG. 13 shows a bacterial encapsulation (BENC) system. In this example, a B. thuringiensis sporulation mutant spo0A- (and thus trapped in stationary phase) does not contain any crystals (left panel); however, when the sequence of Cry5B is expressed under a stationary phase promoter (such as Cry3A; construct depicted in center panel) in these cells, Cry5B crystals (right panel, arrow) are made and trapped inside the bacterium (right panel).

FIG. 14 shows that B. thuringiensis spo0A- cells expressing Cry5B are bioactive against C. elegans, whether treated or not with carvacrol for 15 or 60 minutes.

FIGS. 15A-C shows the efficacy of Bacillus thuringiensis (Bt) spore-crystal lysate (SCL) against hookworms. FIG. 15A depicts life cycle of Bt and production of SCL. FIG. 15B depicts single dose Cry5B SCL from Bt eliminates A. ceylanicum hookworm adults in hamsters in vivo; FIG. 15C depicts single dose Cry5B SCL from Bt eliminates fecal egg counts from hookworm-infected hamsters in vivo.

FIGS. 16A-B shows the effects of irradiation on Bt SCL expressing Cry5B. The spores are largely, although not completely, inactivated but the Cry5B protein is no longer active. FIG. 16A depicts effects of 15 kGy of gamma irradiation on spore counts for two different Cry- Bt strains (4.D.8, 4.D.9) transformed with a Cry5B expressing plasmid or (4.D.8 only) empty vector. FIG. 16B depicts effects of 15 kGy gamma irradiation on Cry5B efficacy expressed in 4.D.8 and 4.D.9 against C. elegans at two doses. N F/D=not freeze-dried SCL; F/D=freeze-dried SCL; 15 kGy=freeze-dried SCL subjected to 15 kGy of irradiation.

FIGS. 17A-F shows the anti-nematode efficacy of BaCC (live spo0A- Bt cells expressing cytosolic Cry5B crystals). FIG. 17A depicts dose-dependent toxicity curves with BaCC cells either fed intact or broken open prior to feeding (by bead beating). FIG. 25B depicts effects on hookworm burdens of Cry5B expressed in SCL and BaCC cells in vivo against A. ceylanicum infections in hamsters. Control includes spo0A- empty-vector cells (Ba), For comparison, the efficacy of SCL is shown. FIG. 17C depicts effects of the same BaCC strains on hookworm fecal egg counts. FIG. 17D depicts the sporulation-defective Bt with a crystal protein trapped in the cytosol. FIG. 17E depicts C. elegans intoxicated by BaCC in comparison to empty Spo0A- Bt cells. FIG. 17F depicts a dot plot showing effects of increasing concentrations of Cry5B-BaCC on C. elegans brood size.

FIGS. 18A-G shows that IBaCC is a potent anthelmintic in vivo. FIG. 18A depicts the IBaCC strategy. Bacillus thuringiensis lacking the spo0A gene cannot sporulate but rather stays in the stationary phase. FIG. 18B depicts a bar graph showing the colony forming units of Spo0A- Bt expressing Cry5B untreated and treated with carvacrol. FIG. 18C depicts a polyacrylamide gel stained with coomassie blue showing the expression of Cry5B in Spo0A- Bt untreated and treated with carvacrol. FIG. 18D depicts a scatter dot plot showing the A. ceylanicum burden in IBaCC treated mice. FIG. 18E depicts a scatter dot plot showing the effects of IBaCC treatment on fecal egg counts in mice. FIG. 18 depicts a scatter dot plot showing the A. ceylanicum burden in mice treated with a placebo, BaCC-Cry5B or IBaCC-Cry5B. FIG. 18G depicts a plot showing fecal egg counts in mice treated with a placebo, BaCC-Cry5B, or IBaCC-Cry5B. Note, for both SCL (see FIG. 17), BaCC (FIGS. 17, 18), and IBaCC (see FIG. 18), efficacy of the total bacterial product is superior to that of purified protein (FIG. 8, FIG. 19A, B) against the same hookworm infections in hamsters. Thus IBaCC preserves the efficacy and advantages of expressing Cry5B in Bt, which is superior to purified Cry5B protein in efficacy, while IBaCC also preserves the advantage and complete safety of purified protein by killing the bacterium.

FIGS. 19A-F shows that pre-neutralization of stomach acid with enhances the efficacy of Cry5B treatment in vivo. FIG. 19A depicts a scatter dot plot showing A. ceylanicum burden in hamsters treated with Cry5B purified protein at a dose of 8 mg/kg with or without pre-treatment with stomach acid neutralizing agents, cimetidine and NaHCO₃. FIG. 19B depicts a scatter dot plot showing fecal egg counts in hamsters treated with Cry5B purified protein at a dose of 8 mg/kg with or without pre-treatment with stomach acid neutralizing agents, cimetidine and NaHCO₃. FIG. 19C depicts a scatter dot plot showing A. ceylanicum burden in hamsters treated with increasing concentrations of SCL-Cry5B with or without pre-treatment with the stomach acid neutralizing agent cimetidine. FIG. 19D depicts a scatter dot plot showing fecal egg counts in hamsters treated with increasing concentrations of SCL-Cry5B with or without pre-treatment with the stomach acid neutralizing agent cimetidine. FIG. 19E depicts a scatter dot plot showing A. ceylanicum burden in hamsters treated with increasing concentrations of IBaCC-Cry5B with or without pre-treatment with the stomach acid neutralizing agent cimetidine. FIG. 19F depicts a scatter dot plot showing fecal egg counts in hamsters treated with increasing concentrations of IBaCC-Cry5B with or without pre-treatment with the stomach acid neutralizing agent cimetidine.

FIG. 20 shows that IBaCC-Cry5B is effective against L3 intestinal Ascaris suum in STAT6-/-mice. A scatter dot plot shows A. suum burden in mice treated with Cry5B-IBaCC with or without pre-treatment with cimetidine.

FIGS. 21A-B shows that freeze-dried Cry5B-IBaCC retains full bioactivity against hookworm infections in vivo. FIG. 21A depicts a scatter dot plot showing A. ceylanicum burden in hamsters treated with a water control, Cry5B-IBaCC, or Cry5B-IBaCC freeze-dried (FD). FIG. 21B depicts a scatter dot plot showing fecal egg counts in hamsters treated with a water control, Cry5B-IBaCC, or Cry5B-IBaCC freeze-dried (FD).

FIGS. 22A-D shows that Cry5B-IBaCC is effective against Necator americanus. FIG. 22A depicts a scatter dot plot showing N. americanus burden in hamsters treated with Cry5B-SCL or a water control. FIG. 22B depicts a scatter dot plot showing fecal egg counts in hamsters treated with Cry5B-SCL or a water control FIG. 22C depicts a scatter dot plot showing N. americanus burden in hamsters treated with Cry5B-IBaCC, freeze-dried (FD) Cry5B-IBaCC, or a water control. FIG. 22D depicts a scatter dot plot showing fecal egg counts in hamsters treated with Cry5B-IBaCC, freeze-dried (FD) Cry5B-IBaCC, or a water control. Since hamsters infected with Necator are heavily immunosuppressed with daily dexamethasone treatment, the data also show an intact immune system is not needed for Cry5B activity.

FIGS. 23A-B shows that the immune system is not required for Cry5B efficacy in vivo. FIG. 23A depicts a scatter dot plot showing fecal egg counts in immuno-suppressed mice treated with Cry5B-SCL. FIG. 23B depicts a scatter dot plot showing H. polygyrus burden in immuno-suppressed mice treated with Cry5B-SCL.

FIGS. 24A-C shows that Cry5B treatment will remain effective with repeated doses. FIG. 24A depicts a scatter dot plot showing A. ceylanicum burden in hamsters treated with a water control or purified Cry5B with or without pre-treatment with Cry5B. FIG. 24B depicts a scatter dot plot showing fecal egg counts in hamsters treated with a water control or purified Cry5B with or without pre-treatment with Cry5B FIG. 32C depicts a scatter dot plot showing A. ceylanicum burden in hamsters treated with a water control, purified Cry5B or Cry5B-SCL with or without pre-treatment with Cry5B-SCL, and with or without pretreatment with spores.

FIGS. 25A-B shows bioactivity of carvacrol-treated Crystal-E. coli on C. elegans. FIG. 25A depicts a bar graph measuring colony-forming units (CFUs) of carvacrol (1 mg/mL) treated E. coli cells harboring nematicidal Crystal protein gene or empty vector control. FIG. 25B depicts a bar graph measuring the bioactivity of E. coli cells harboring nematicidal Crystal protein (Crystal- E. coli) gene or empty vector (Vector- E. coli) on the nematode Caenorhabditis elegans. The concentration of both Crystal-E. coli and Vector-E. coli is 1.8×10⁷ cell/mL in the C. elegans assays. Worms (n=10 per condition) were incubated at 25° C. for 16 hr. NT=not treated with carvacrol; T=treated with carvacrol.

DETAILED DESCRIPTION

Disclosed are methods of treating or preventing STH infection by administering to a subject a preparation of killed or inactive bacteria recombinantly expressing a nematicidal protein (e.g., crystal protein from Bacillus thuringiensis) in the cytosol of the bacterium. Such recombinant bacteria are treated with an anti-microbial agent such that the bacteria are killed before or during administration. In these particular methods, because the bacteria are dead when administered, any bacterium, including non-food grade bacteria, can be administered to a subject to treat an STH infection.

Microbes

In certain embodiments, the bacteria of the invention are non-sporulating bacteria. As used herein, the term “non-sporulating bacterium” includes wild-type bacteria that are incapable of producing spores (e.g., certain Gram-negative bacteria) as well as genetic variants of spore-forming bacteria that have been engineered to be defective in sporulation (e.g., certain Gram-positive bacteria). As used herein, unless the context makes clear otherwise, “a mutation resulting in a defect in sporulation” or “a genetic mutation that results in a defect in sporulation” refers to any genetic mutation that results in a defect in a member of the sporulation pathway and/or any genetic mutation that prevents the formation of viable spores.

In some embodiments, sporulation-deficient bacteria are advantageous. An example of a sporulation deficient bacterium is a spo0A- Bacillus thuringiensis. Any mutation or combination of mutations that confers sporulation deficiency but that does not substantially affect viability or heterologous gene expression can be used. These mutations include but are not limited to mutations in the following genes: kinA, kinB, spo0A, spo0B, spo0E, spo0F, spo0J, spo0M spoIIB, spoIID, spoIIE, spoIIF, spoIIG, spoIIL, spoIIM, spoIIIA, spoIIIB, spoIIIE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK. (Silvaggi, J., et al. Unmasking novel sporulation genes in Bacillus subtillus. J Bacteriol. 186, 8089-8095, 2004; Sandman, K., et al. Genetic Analysis of Bacillus subtilis spo Mutations Generated by Tn917-Mediated Insertional Mutagenesis. Genetics. 117, 603-617, 1987; Malvar and Baum, Tn5401 Disruption of the spoOF Gene, Identified by Direct Chromosomal Sequencing, Results in CrylIlIA Overproduction in Bacillus thuringiensis. J Bacteriol. 176, 4750-4753, 1994)

Bacteria are particularly applicable to the control of STHs because 1) recombinant bacteria can cheaply express large amounts of Cry proteins prior to administration into the GI tract of a mammalian subject, and Cry proteins so expressed, independent of any Cry proteins that may be secreted by bacteria in the GI tract, have been shown to have a significant impact on STHs, 2) studies using purified Cry protein to treat hookworms, whipworms, and H. bakeri, all in infected rodents, demonstrate that STHs in the mammalian GI tract can ingest and be killed/intoxicated by Cry proteins, 3) recombinant bacteria expressing a therapeutic protein, in which the protein is not purified, are cheaper to produce since no purified protein is needed, and 4) recombinant bacteria delivering STH curing proteins (e.g., Cry5B) are more effective that purified proteins (e.g., Cry5B) at the same bio-active protein dose (e.g., total Cry5B) in curing infections.

Microbes of the disclosed compositions and methods include killed and inactivated forms of Bacillus sp., including Bacillus subtilis (e.g., Bacillus subtilis natto, and Bacillus subtilis PY79), B. cereus, (e.g., B. cereus var. Toyoi (Toyocerin), B. cereus var. toyoii), B. toyonensis, B. clausii, B. pumilus and Bacillus thuringiensis. Bacillus subtilis has been extensively characterized as a safely ingested food additive in humans (see Example 14, infra, references 15-27). In certain exemplary embodiments, killed and inactive forms of Bacillus thuringiensis are used.

Other useful bacteria include but are not limited to non-sporulating variants of Lactococcus sp., Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Clostridium sp., Sporolactobacillus sp, Sporosarcina sp., Brevibacillus sp, Leuconostoc sp., Pedicoccus sp., Enterococcus sp. and Escherichia sp. Lactococcus sp. includes but is not limited to L. lactis. Lactobacillus sp. includes but is not limited to L. casei, L. paracasei, L. acidophilus, L. bulgaricus, L. delbrueckii subsp. bulgaricus, L. helveticus, L. plantarum, L. salivarius, L. reuteri, L. gasseri, and L. animalis. Bifidobacterium sp. includes but is not limited to B. animalis, B. bifidum, B. breve, B. infantis, and B. longum. Streptococcus sp. includes but is not limited to S. thermophilus. Clostridium sp. includes but is not limited to Clostridium butyricum. Sporolactobacillus sp. includes but is not limited to Sporolactobacillus vineae. Sporosarcina sp. includes but is not limited to Sporosarcina pasteurii. Brevibacillus sp. includes but is not limited to Brevibacillus laterosporus.

Still other useful bacteria useful in connection with the claimed invention include killed and inactivated forms of Gram-negative bacteria. In certain exemplary embodiments, the Gram-negative bacteria include E. coli species (e.g., NISSLE 1917) and Pseudomonas species (e.g., Pseudomonas fluorescens). Exemplary Cry-expressing Gram-negative bacteria which can be killed or inactivated by the methods of the invention include the Cry-expressing E. coli strain of Ge et al. (“Hyperexpression of a Bacillus thuringiensis delta-endotoxin-encoding gene in Escherichia coli: properties of the product”, Gene, 93: 49-54 (1990)) and the P. fluorescens strain of Peng et al. (“A Delta-endotoxin encoded in Pseudomonas flurescens displays a high degree of insecticidal activity”, App. Microbiol Biotech., (2003), 63:300-306).

Nematicial Proteins

As used herein, unless the context makes clear otherwise, “nematicidal protein” refers to any protein that has toxic activity against nematodes or helminthes. Exemplary nematicidal proteins include crystal proteins such as the anthelmintic Cry proteins (e.g., Crickmore et al., 1998 Microbiology and Molecular Biology Reviews 62(3): 807-813; Schnepf et al., 1998 Microbiology and Molecular Biology Reviews 62(3): 775-806; including but not limited to the B. thuringiensis Cry proteins Cry5B (e.g., SEQ ID NO:1) and its subvariants, Cry13A (e.g., SEQ ID NO:2) and its subvariants, Cry14A (e.g., SEQ ID NO:3) and its subvariants, Cry21A (e.g., SEQ ID NOS:4-5) and its subvariants, and Cry6A and its subvariants (e.g., SEQ ID NO:6)) in the bacterium for delivery into a helminth (e.g., roundworm)-infected vertebrate animal gastrointestinal tract via oral dosing (gavage, drinking, eating, pill, capsule, powder, etc.). The Cry proteins are expressed in the cytosol of the bacterium, allowing access to the anthelmintic protein after the bacterium lyses or opens up either due to digestion within the gastrointestinal tractingestion and digestion of bacteria by the parasitic helminths (e.g., roundworms such as hookworms, whipworms, Ascaris, Strongyloides, veterinary parasitic roundworms of the intestine), etc.

In certain embodiments, a bacterium as provided herein may be introduced that expresses an individual Cry protein or that simultaneously expresses multiple Cry proteins. In some embodiments, multiple bacteria may be introduced, each of which expresses either a different individual Cry protein or simultaneously expresses multiple Cry proteins. In these and related embodiments, it is contemplated that the GI tract may be seeded with bacteria that express either one Cry protein or multiple Cry proteins at the same time. For example, due to the lack of cross-resistance between Cry5B-resistant roundworms and Cry21A-resistant roundworms, simultaneous administration of Cry5B and Cry21A in the gastrointestinal tract may inhibit the development of parasite resistance to the combination therapy.

In the long run, removing antibiotic selection capability (e.g., genetic selection markers) from the plasmids that are used to introduce heterologous Cry protein-encoding sequences, as well as using bacterial strains that are unable to replicate outside the vertebrate host, may be desirable in order to environmentally contain the genetically modified bacteria. For example, LAB (Lactic Acid Bacteria) have been engineered to be autotrophic in thymidine or thymine synthesis such that they can only grow in the vertebrate intestine where thymidine or thymine is present and not in the environment where thymidine or thymine is not present. See, e.g., Steidler L, et al. “Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10.” Nat Biotechnol 21: 785-789 (2003).

Cry-transformed bacteria such as Bacilli or LAB may be cultured and expression of intracellular, membrane-anchored, or secreted Cry protein by such bacteria may be confirmed using antibodies raised against each Cry protein and standard Western blotting or ELISA techniques.

To assess the bioactivity of all constructs, recombinant expressing Cry protein (full length, truncated, or variants) may be fed to the free-living nematode, C. elegans. Cry protein toxicity on C. elegans using LC50, brood-size, developmental inhibition assays on solid media and in liquid wells may then be quantitated. C. elegans can access the Cry proteins either via protein secreted onto the solid media/into the liquid well or by their ability to grind, open and digest bacteria. Confirmation that the recombinant bacteria are making bioactive Cry proteins may be obtained. Furthermore, the bioactivity (e.g., LC₅₀ in μg/mL) may be quantified and the constructs giving the highest activity determined.

Truncations, Variants, and Sub-Variants

The crystal proteins may be truncated to enhance their effectiveness. The usefulness of Bt toxins (e.g., crystal proteins) for controlling STHs may be limited by the protein size that STHs can ingest. Some parasitic roundworms poorly ingest proteins larger than about 40 kD. Thus, the effectiveness of any particular Bt toxin may be limited by size exclusion of proteins that STHs take in and so should be small enough to be readily absorbed by the STH gut while retaining toxic activity. A truncated toxin may be easier to express in bacteria. Producing a truncated toxin also alleviates the requirement that the target STH has the proper proteases present to correctly process full length protoxin (which is inactive) to a truncated, active toxin form. Thus, a truncated toxin is immediately available for intoxication independent of whether the proper protease processing enzymes are present in the STH target. Truncated toxin may also express at a higher level in microbes because truncated toxins are soluble and less likely to form insoluble inclusions in the cell expressing them, which could be toxic to the cell or which could make the toxin fold incorrectly. Accordingly, it is desirable to produce truncated Bt toxin fragments (e.g., crystal protein fragments). Moreover, fragments of certain Bt toxins have been tested and shown to retain toxic activity and have improved biological properties. By “truncated,” when referring to a Bt toxin protein (crystal protein) is meant a Bt toxin protein that is not full-length but retains at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the toxic activity of a corresponding full-length Bt toxin protein.

“Variants” or “subvariants” of Cry proteins include polypeptides with one or more substitutions, e.g., no more than 20 substitutions, alternatively no more than 10 substitutions, or substitutions at 10% or fewer of the residues, relative to a corresponding wild-type polypeptide or truncated version thereof. The variant, subvariant, or truncated polypeptide has at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the activity, e.g., toxic activity, of the corresponding wild-type polypeptide or truncated version. Conservative substitutions include substitutions within the following groups: glycine, alanine, threonine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, cysteine; lysine, arginine; aspartic acid, glutamic acid; serine, threonine; asparagine, glutamine; phenylalanine, tyrosine.

The crystal proteins may be full length, truncated, variants, or subvariants. The truncated crystal protein may include any truncation of the N- and C-termini that still retains toxin activity. The truncated form is not full-length but retains at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the toxic activity of a corresponding full-length Bt toxin protein. For example, the truncated portion may be truncated between the end of conserved block 5 and the C-terminus of the full length protein.

In one embodiment, the truncated crystal protein may contain the toxin domain of the crystal protein and optionally include up to 5, 10, or 20 additional amino acids. The truncated crystal protein may be truncated after a conserved amino acid sequence of block 5 and optionally include up to 5, 10, or 20 additional amino acids. The conserved amino acid sequence of block 5 may contain the motif DRIEF (SEQ ID NO: 23), DRLEF (SEQ ID NO: 24), or some other related sequence as well as surrounding amino acid residues, e.g., three amino acids upstream and two amino acids downstream of this motif. Table 1 shows the block 5 sequences for various Cry proteins. See e.g., Schnepf, E., et al., Bacillus thuringiensis and Its Pesticidal Crystal Proteins, Microbiology and Molecular Biology Reviews 62(3): 775-806, (e.g., at p. 781, FIG. 3) (September 1998); and Crickmore et al., Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins, Microbiology and Molecular Biology Reviews 62(3): 807-813 (September 1998). The truncated crystal protein may also be truncated at the N-terminus. For example, the truncated crystal protein may not contain the first about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids at the N-terminus.

Cry protein variants can exhibit at least 50, 55, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent amino acid sequence identity to a known Cry protein sequence such as any that are disclosed in Crickmore et al., 1998 Microbiology and Molecular Biology Reviews 62(3): 807-813, or in Schnepf et al., 1998 Microbiology and Molecular Biology Reviews 62(3): 775-806, including full length Cry proteins and truncated Cry proteins, Cry protein variants or subvariants thereof. Also contemplated according to certain embodiments are polynucleotides encoding such Cry proteins and truncations and variants thereof.

TABLE 1 Protein Block 5 Conserved Group Cry1A VYIDRIEFVP (SEQ ID NO: 7) Cry3A VYIDKIEFIP (SEQ ID NO: 8) Cry4A VLIDKIEFLP (SEQ ID NO: 9) Cry5A VFLDRIEFIP (SEQ ID NO: 10) Cry5B LFLDRIEFVP (SEQ ID NO: 11) Cry7A FYVDSIEFIP (SEQ ID NO: 12) Cry8A VYIDRIEFIP (SEQ ID NO: 13) Cry9A VYVDRIEFIP (SEQ ID NO: 14) Cry10A IYIDKIEFIP (SEQ ID NO: 15) Cry12A MVLDRIEFVP (SEQ ID NO: 16) Cry13A IYLDRLEFVP (SEQ ID NO: 17) Cry14A IFIDRIEFIP (SEQ ID NO: 18) Cry19A LILDKIEFLP (SEQ ID NO: 19) Cry20A FVLDKIELIP (SEQ ID NO: 20) Cry21A LFLDRIEFIS (SEQ ID NO: 21) Consensus i-iDkIEFiP (SEQ ID NO: 22)

In Table 1, the consensus sequence denotes the positions at which at least 75% of the aligned proteins in the group have an identical or conserved amino acid sequence. An uppercase letter in the sequence indicates that at least 75% of the residues at that position are identical. A lowercase letter indicates that at least 75% of the residues at that position are conserved. Conserved amino acids fall into the following groups: a (A, G, S, T, or P); d (D, E, N, or Q); f (F, W, or Y)l I (I, L, M, or V), and k (K or R).

The truncated crystal protein may be a truncated form of Cry5B such as B. thuringiensis Cry5B (FIG. 2). Truncated Cry5B may extend from about amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to at least about amino acid 693. The truncated form of Cry5B may optionally include up to an additional 5, 10, 20, 30, 40, or 50 amino acids from the C-terminus after conserved block 5, e.g., through about 698, 703, 713, 723, 733, or 743.

The truncated crystal protein may be a truncated form of Cry13A such as B. thuringiensis Cry13A (FIG. 3). Truncated Cry13A may extend from about amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to at least about amino acid 688. The truncated form of Cry13A may optionally include up to an additional 5, 10, 20, 30, 40, or 50 amino acids from the C-terminus after conserved block 5, e.g., through about 693, 698, 708, 718, 728, or 738.

The truncated crystal protein may be a truncated form of B. thuringiensis Cry14A (FIG. 4). Truncated Cry14A may extend from about amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to at least about amino acid 675. The truncated form of Cry14A may optionally include up to an additional 5, 10, 20, 30, 40, or 50 amino acids from the C-terminus after conserved block 5, e.g., through about 680, 685, 695, 705, 715, or 725.

The truncated crystal protein may be a truncated form of Cry21A such as B. thuringiensis Cry21Aa1 (FIG. 5A) or Cry21Aa2 (FIG. 5B). Truncated Cry21A may extend from about amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to at least about amino acid 685. The truncated form of Cry21A may optionally include up to an additional 5, 10, 20, 30, 40, or 50 amino acids from the C-terminus after conserved block 5, e.g., through about 690, 695, 705, 715, 725, or 735.

Nucleic acid molecules encoding amino acid sequence variants, truncated versions, or both, of a Cry protein are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by, for example, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of protein. Moreover, the invention includes synthetic nucleic acid molecules where nucleotides are modified to include codons preferred in a particular organism, remove codons rarely used in a particular organism, or remove sequences that may inhibit transcription or RNA processing and the like.

Cry protein truncations may at least include conserved blocks 1-5. As seen in FIGS. 1A and 1B, alignment of known Cry toxins reveals five conserved sequence blocks (blocks 1-5) that are common to a majority of the proteins and are thought to be located in the active toxin domain. See de Maagd, R. A., et al. “How Bacillus thuringiensis has evolved specific toxins to colonize the insect world.” TRENDS IN GENETICS 17(4): 193-99 (April 2001). Comparison of the carboxy-terminal halves of the sequences have suggested the presence of three additional blocks that lie outside of the active toxic core. See Schnepf, E., et al. “Bacillus thuringiensis and Its Pesticidal Crystal Proteins.” MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS 62(3): 775-806 (September 1998). Thus, Cry protein truncations may be truncated after the conserved amino acid sequence of block 5 (e.g., DRIEF (SEQ ID NO: 23) or DRLEF (SEQ ID NO: 24)). Alternatively, Cry protein truncations may be truncated after the conserved amino acid sequence of block 5 (e.g., DRIEF (SEQ ID NO: 23) or DRLEF (SEQ ID NO: 24)) plus an additional about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of the c-terminal domain.

The complete amino acid sequence of Cry5Ba1 is listed in FIG. 2. The conserved amino acid sequence DRIEF (SEQ ID NO: 23) in Cry5B ends at amino acid number 693. Thus, a truncated form of Cry5B may include at least amino acids 50 through about 693. A truncated form of Cry5B may extend from about amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to at least about amino acid 693. Alternatively or in addition to, a truncated form of Cry5B may include about 5, 10, 15, 20, 25, 30, 35, or 40 additional amino acids of the c-terminal domain.

The complete amino acid sequence of Cry13Aa1 is listed in FIG. 3. The conserved amino acid sequence DRLEF (SEQ ID NO: 24) in Cry13A ends at amino acid number 688. Thus, a truncated form of Cry13A may include at least amino acids 50 through about 688. A truncated form of Cry5B may extend from about amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to at least about amino acid 688. Alternatively or in addition to, a truncated form of Cry13A may include about 5, 10, 15, 20, 25, 30, 35, or 40 additional amino acids of the c-terminal domain.

The complete amino acid sequence of Cry14Aa1 is listed in FIG. 4. The conserved amino acid sequence DRIEF (SEQ ID NO: 23) in Cry14A ends at amino acid number 675. Thus, a truncated form of Cry14A may include at least amino acids 50 through about 675. A truncated form of Cry5B may extend from about amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to at least about amino acid 675. Alternatively or in addition to, a truncated form of Cry14A may include about 5, 10, 15, 20, 25, 30, 35, or 40 additional amino acids of the c-terminal domain.

The complete amino acid sequence of Cry21Aa1 and Cry21Aa2 are listed in FIGS. 5A and 5B, respectively. The amino acid sequence of Cry21Aa2 is about 98% identical to the sequence of Cry21Aa1. The conserved amino acid sequence DRIEF (SEQ ID NO: 23) in Cry21A ends at amino acid number 685. Thus, a truncated form of Cry21A may include at least amino acids 50 through about 685. A truncated form of Cry5B may extend from about amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to at least about amino acid 685. Alternatively or in addition to, a truncated form of Cry21A may include about 5, 10, 15, 20, 25, 30, 35, or 40 additional amino acids of the c-terminal domain.

Anthelmintic Experiments

Once heterologous Cry protein expression and bioactivity are confirmed in a desired bacterium, the modified bacteria may be used for curative-type and preventative-type anthelmintic experiments.

Antibody production: Antibodies against recombinant Cry proteins (e.g., Cry5B, Cry21A, Cry14A, Cry13A, and Cry6A, full length and truncated proteins) may be produced and purified according to standard methodologies (e.g., Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009).

Bioactivity tests: To assess the bioactivity of all constructs, recombinant bacilli or other bacteria expressing heterologous Cry proteins are fed to the free-living nematode, C. elegans. C. elegans can access the Cry proteins either via protein secreted onto the solid media/into the liquid well, by protein naturally released as bacteria break open, or by their ability to grind and digest bacteria to open the bacterial cells.

Rodent and parasite tests: Three intestinal parasitic nematodes—H. bakeri (small intestine nematode parasite) in mice, and Trichuris muris (whipworm) in mice, and A. ceylanicum (hookworm) in hamsters are tested. The tests address: 1) where in the GI tract do heterologous Cry-expressing bacteria reside and for how long; and 2) how do these bacteria affect the acquisition and progression of intestinal nematode parasites.

Parasite tests: Naïve (uninfected) mice are gavaged with the best heterologous Cry-protein expressing recombinant bacterial strain(s) based on expression and bioactivity.

Protect against progression test: Mice are infected with H. bakeri. Two weeks later, infected mice are treated with heterologous Cry-protein expressing or control bacteria, respectively. Intestinal worm burdens and fecal egg counts are used to determine if the recombinant bacteria provide anthelmintic therapy in mice with pre-existing nematode infections.

Exemplary Parasites

The disclosed methods relate to the control of parasitic worms, e.g., nematodes and platyhelminths, using crystal proteins from Bacillus and their derivatives. Parasitic worms within the scope of the invention include but are not limited to those in Class Adenophorea, e.g., Order Mononchida, Family Plectidae, and Order Stichosomida, Family Mermithidae and Tetradonematidae; Class Secernentea, e.g., Order Rhabditida, Family Carabonematidae, Cephalobidae, Chambersiellidae, Heterorhabditidae, Oxyuridae, Panagrolaimidae, Rhabditidae, Steinernematidae, Syrphonematidae, Syrphonematidae, or Thelastomatidae; Order Spirurida, Family Filariidae, Onchocercidae, Physalopteridae, Syngamidae, Spiruridae, Subuluridae, or Thelaziidae; Order Diplogasterida, Family Diplogasteridae; and Order Tylenchida, Family Allantonematidae, Aphelenchidae, Aphelenchoididae, Entaphelenchidae, Fergusobiidae, Phaenopsitylenchidae, Sphaerulariidae, Anguinidae, Dolichodoridae, Belonolaimidae, Pratylenchidae, Hoplolamidae, Heteroderidae, Criconematidae, Tylenchulidae or Tylenehidae. In one embodiment, the parasite is from Class Secernentea, Order Ascaridida, Family Ascarididae; Class Adenophorea, Order Trichurida, Family Trichuridae; Class Secernentea, Order Strongylida, Family Ancylostomatidae (ancylostomidae) or Trichostrongylidae; or Class Secernentea, Order Spirurida, Family Dracunculidae, Filariidae, or Onchocercidae.

The parasite may be a helminth. Helminths within the scope of the invention include but are not limited to those from Phylum Annelida, Class Polychaetae, Class Myzostomida, Class Clitellata, Subclass Hirudinea, Order Gnathobdellidae, Order Rhynchobdellidae; Phylum Platyhelminthes (Flatworms), Class Turbellaria, Class Monogenea, Order Monopisthocotylea, Order Polyopisthocotylea, Class Trematoda, Subclass Aspidogasrea, Subclass Digenea; Super Order Anepitheliocystida, Order Strigeatida, Family Schistosomatidae, Subfamily Schistosomatinae, Genus Schistosoma, Order Echinostomatida, Family Fasciolidae, Family Paramphistomatidae, Family Echinostomatidae; Super Order Epitheliocystida, Order Plagiorchiida, Family Dicrocoeliidae, Family Troglotrematidae, Order Opisthorchiida, Family Heterophyidae, Family Opisthorchiidae, Class Cestoda, Subclass Cestodaria, Subclass Eucestoda, Order Pseudophyllidea, Family Diphyllobothriidae, Order Cyclophyllidea, Family Taeniidae, Family Hymenolepididae, Family Dilepididae, Family Mesocestoididae, Order Tetraphyllidea, Order Proteocephalata, or Order Spatheobothridea. For example, Cry proteins with the scope of the invention may be employed to prevent, inhibit or treat Roundworm, Whipworm, Hookworm, Schistosome, or Trematodes.

The parasite may also be gastrointestinal tract parasitic roundworms/nematodes. The gastrointestinal tract parasitic roundworms/nematodes may include but are not limited to the following species: Haemonochus, Cooperia, Ostertagia, Trichostrongylus, Teladorsagia, Nematodirus, Ancylostoma, Cyathostominea/Cyathostomin/Cyathostome, Strongylus, Parascaris, Ascaris, Trichuris, Oesophagostomum/Oesophagustomum, Trichiuris, Bunostomum, Oxyuris, Chabertia, Habronema, Draschia, Triodontophorus, Toxocara, Toxascaris, and Uncinaria. Haemonochus species includes but is not limited to Haemonchus contortus and Haemonchus placei, Cooperia species includes but is not limited to Cooperia oncophora, Cooperia pectinata, and Cooperia curticei. Ostertagia species includes but is not limited to Ostertagia ostertagi, Ostertagia (Teladorsagia) circumcincta, and Ostertagia trifurcate. Trichostrongylus species includes but is not limited to Trichostrongylus axei, Trichostrongylus colubriformis, and T. circumcincta. Teladorsagia species includes but is not limited to Teladorsagia (Ostertagia) circumcincta. Nematodirus species includes but is not limited to Nematodirus spathiger. Ancylostoma species includes but is not limited to Ancylostoma caninum, Ancylostoma braziliense, and Ancylostoma tubaeforme. Cyathostominea/Cyathostomin/Cyathostome nematodes are also included. Strongylus species (small and large) includes but is not limited to Strongylus vulgaris, Strongylus equinus, and Strongylus edentatus. Parascaris species includes but is not limited to Parascaris equorum. Strongyloides species includes but is not limited to Strongyloides westeri. Ascaris species includes but is not limited to Ascaris suum. Trichuris species includes but is not limited to Trichuris globulosa, Trichuris suis, Trichuris campanula, and Trichuris vulpis. Oesophagostomum/Oesophagustomum species includes but is not limited to Oesophagustomum dentatum, Oesophagustomum quadrispinulatum, Oesophagostomum columbianum, and Oesophagostomum venulosum. Trichiuris species includes but is not limited to Trichiuris ovis. Bunostomum species includes but is not limited to Bunostomum trigonocephalum. Oxyuris species includes but is not limited to Oxyuris equi (pin worms). Chabertia species includes but is not limited to Chabertia ovina. Habronema species includes but is not limited to Habronema microstoma and Habronema muscae. Draschia species includes but is not limited to Draschia megastoma. Triodontophorus species includes but is not limted to Triodontophorus minor and Triodontophorus serrates. Toxocara species includes but is not limted to Toxocara canis and Toxocara cati. Toxascaris species includes but is not limted to Toxascaris leonine. Uncinaria species includes but is not limted to Uncinaria stenocephala. Human parasitic roundworms of the gastrointestinal tract include but are not limited to the hookworms Ancylostoma duodenale and Necator americanus, the whipworm Trichuris trichiura, the roundworm Ascaris lumbricoides, the threadworm Strongyloides stercoralis, and the pinworm Enterobius vermiculari.

Anti-Microbial Agents

In the disclosed methods, the recombinant bacteria expressing a crystal protein can be treated with an anti-microbial agents. Anti-microbial agents can be used on the recombinant bacteria before administration to a subject, or concomitant with administration to the subject. An advantage of killing the recombinant bacteria is that otherwise non-food safe bacteria can be used in the disclosed methods. Such non-food safe bacteria, such as Bacillus thuringiensis which is closely related to Bacillus cereus that can cause food poisoning, express very high levels of Cry proteins such as Cry5B and improve the efficacy of the protein when co-administered versus when the protein is administered in a pure form without the bacterium

Suitable anti-microbial agents are those that (1) sufficiently kill the recombinant bacteria; and (2) do not substantially affect the activity and/or levels of the crystal protein. Examples of suitable anti-microbial agents include, but are not limited to, antibiotics (such as a beta-lactam antibiotic), bacteriocidal agents, iodine, terpenes, formaldehyde, and irradiation. Examples of terpenes include, but are not limited to, thymol, eugenol, geraniol, carvacrol, and citral, or a combination thereof. Carvacrol is especially useful.

Additional Therapeutic Agents

In certain embodiments the crystal protein-recombinant bacteria are administered in combination with at least one additional therapeutic agent. This additional agent can be, for example, a bacterium expressing or capable of expressing, a crystal protein, a small molecule, or a polypeptide (including antibodies and fragments thereof). In a further embodiment, the additional therapeutic is a nicotinic acetylcholine receptor agonist. In certain embodiments, the additional therapeutic agent is administered simultaneously with recombinant bacteria. In certain embodiments the additional therapeutic agent is administered sequentially (and in either order) with the recombinant bacterium. In certain embodiments, the nicotinic acetylcholine receptor agonist is from the levamisole family of nicotinic acetylcholine receptor agonists. In certain embodiments, the nicotinic acetylcholine receptor agonist is levamisole. In certain embodiments, the levamisole is administered in an amount of about 0.1 mg/kg to about 5.0 mg/kg. In certain embodiments the nicotinic acetylcholine receptor agonist is pyrantel or tribendimidine. In certain embodiments, the pyrantel is administered in an amount of about 1.0 mg/kg to about 15.0 mg/kg. In certain embodiments, the tribendimidine is administered in an amount of about 0.25 mg/kg to about 10 mg/kg.

Administration, Dosage Forms, Pharmaceutical Compositions

The present invention describes compositions and methods for administration of killed or inactivated bacterial cells to the gastrointestinal tract of a subject. The methods include administering the bacteria in food or as a food supplement. Oral administration is preferably in an aqueous suspension, emulsion, powder or solid. The composition may be formulated into a food or added to food by the user prior to consumption. Administration to the gastrointestinal tract may also be in the form of an anal suppository (e.g., in a gel or semi-solid formulation). All such formulations are made using standard methodologies.

The method is typically practiced on any animal where inhibiting pathogen or parasites is desired. In certain embodiment, the animal is a human. However, the animal can be any livestock or zoological specimen where such inhibition of parasites/pathogens provides economic and health benefits. Any animal can benefit by the claimed methods, including birds, reptiles, mammals such as horses, cows, sheep, goats, pigs, and the like domesticated animals, or any of a variety of animals of zoological interest. Other purposes are readily apparent to one skilled in the arts of nutrient absorption, feed utilization and bioavailability.

The present invention further contemplates a therapeutic system for treating, reducing and/or controlling parasitic infections. Typically, the system is in the form of a package containing a therapeutic composition of the present invention, or in combination with packaging material. The packaging material includes a label or instructions for use of the components of the package. The instructions indicate the contemplated use of the packaged component as described herein for the methods or compositions of the invention. By way of example, and not of limitation, a system can comprise one or more unit dosages of a therapeutic composition according to the present invention. Alternatively, the system can alternately contain bulk quantities of a therapeutic composition. The label contains instructions for using the therapeutic composition in either unit dose or in bulk forms as appropriate, and may also include information regarding storage of the composition, disease indications, dosages, routes and modes of administration and the like information.

Furthermore, depending upon the particular contemplated use, the system may optionally contain either combined or in separate packages one or more of the following components: bifidogenic oligosaccharides, flavorings, carriers, and the like components. One particularly preferred embodiment comprises unit dose packages of bacterial cells for use in combination with a conventional liquid product, together with instructions for combining the bacteria with the formula for use in a therapeutic method.

Different dosage regimens may be used in the disclosed methods. In some embodiments, a daily dosage is administered once, twice, three times, or four times a day for one, two, three, four, five, six, seven, eight, nine, or ten days. In some embodiments, a once- or twice-daily dosage is administered every other day.

Administration of the compositions containing the active ingredients effective in inhibiting parasite growth in the intestine and in feces generally consist of one to ten unit dosages of 10 mg to 10 g per dosage of the composition for one day up to one month for a human of approximately 100 kg body weight. Unit dosages are generally given once every twelve hours and up to once every four hours. Preferably two to four dosages of the composition per day, each comprising about 0.1 g to 50 g per dosage, for one to seven days are sufficient to achieve the desired result.

A preferred method involves the administration into the digestive tract of from 1×10² to 1×10¹⁰ of bacterium per day, in some embodiments from 11×10³ to 1×10⁶, in other embodiments from 1×10⁶ to 1×10⁹, and more preferably about from 5×10⁸ to 1×10⁹ bacterium per day. Exemplary dosages range from about 1×10³ to 1×10⁶ bacterium per day, or alternatively range from about 1×10⁶ to 1×10⁹ bacterium per day.

In various specific embodiments, an effective dose of a composition of the present disclosure can be in a range of from 1.0 gm to 15.0 gm for an adult patient, more preferably between about 2.0 gm and about 10.0 gm of the composition. Effective doses can be administered to a subject at any suitable frequency, e.g., at least once a week, preferably once a day. Pediatric dosages may be in the range of 15% to 90% of adult dosages.

In other embodiments, a constant dosage of the composition can be administered over time, for example about 2 gm to about 4 gm per day, up to about 6 g to about 10 g per day, depending on the severity of the physiological condition. Once the infection has been effectively ameliorated, the subject can in many instances decrease the dosage to about 2 gm to about 4 gm per day for maintenance purposes. The desired dose may be presented in multiple (e.g., two, three, four, five, six, or more) sub-doses administered at appropriate intervals throughout the day.

The pharmaceutical compositions comprising the crystal protein-expressing recombinant bacteria can be administered via any of the accepted modes of administration or agents known in the art. However, oral administration is preferred because this route of delivery delivers the recombinant bacteria to the GI tract. The dosage form can be, for example, a solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, pills, soft elastic or hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols, or the like, and can be in unit dosage forms suitable for simple administration of precise dosages. One exemplary embodiment of the dose form is a capsule containing the composition of the disclosure including the bacterial species in a dried form, blended with pharmaceutical carrier. The capsule for such dose form can be of any suitable type, e.g., a gelatin capsule of a conventional variety.

The physiologically compatible carrier medium with which the bacterial species are employed, can be of any simple type, e.g., a pharmaceutically acceptable carrier such as fructo-oligo-saccharide (FOS) medium, or other soluble fiber, sugar, nutrient or base material for the composition, with which the bacterial species can be formulated, e.g., in an orally administrable form. Other carrier media include mannitol, inulin (a polysaccharide), polydextrose, arabinogalactan, polyolslactulose, lactitol, etc. A wide variety of materials can be used as carrier material in the practice of the present disclosure, as will be apparent to those of ordinary skill in the art, based on the description herein.

The carrier medium, when present, can be blended with the bacterial species in any suitable amounts, such as an amount of from 5% to 95% by weight of carrier medium, based on the total weight of the bacterial species and the carrier medium, in various embodiments. In other embodiments, the amount of carrier medium may be in a range having a lower limit of any of 5%, 10%, 12%, 15%, 20%, 25%, 28%, 30%, 40%, 50%, 60%, 70% or 75%, and an upper limit, higher than the lower limit, of any of 20%, 22%, 25%, 28%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 95%. The amount of carrier medium in a specific embodiment may be determined based on considerations of the specific dose form, relative amounts of the bacterial species, the total weight of the composition including the carrier medium and the bacterial species, and the physical and chemical properties of the carrier medium, and other factors, as known to those of ordinary skill in the probiotic formulation art.

In certain embodiments, the bacterial cells are formulated in a composition that protects the cells and/or Cry proteins from the acid environment of the stomach. Accordingly, the invention includes a composition containing a bacterium and a pharmaceutically-acceptable acid-resistant (“enteric”) carrier. By acid-resistant is meant that the carrier or coating does not dissolve in an acidic environment. An acidic environment is characterized by a pH of less than 7. The acid-resistant carrier is resistant to acids at pH less than about 4.0. Preferably, the carrier does not dissolve in pH 2-3. Most preferably, it does not dissolve in pH of less than 2. To protect bacterial cells from stomach acids, the cells are coated or encapsulated with the acid-resistant carrier.

In certain embodiments, the coating is pH-sensitive. For example, the coating may dissolve after the pH is greater than 4.0. For example, the coating dissolves in a neutral environment as is encountered in the small intestine, and does not dissolve in an acidic environment as is encountered in the stomach. Alternatively, the enteric coating dissolves when exposed to specific metabolic event such as an encounter with a digestive enzyme that is found in the small intestine. For example, the coating is digested by a pancreatic enzyme such as trypsin, chymotrypsin, or a pancreatic lipase. The formulation is hydrated in the small intestine. Digestion or dissolution of the coating allows liberation of bacterial cells, e.g., Bacillus cells, into the intestine.

In other embodiments, bacterial cells are stabilized in a gel or paste such as an anhydrous carbohdrate paste. In alternate formulations, the cells are lyophillized and/or suspended in a gel or paste. Enteric coating materials are known in the art, e.g., malic acid-propane 1,2-diol. Cellulose derivatives, e.g., cellulose acetate phthalate or hydroxypropyl methylcellulose phthalate (HPMCP), are also useful in enteric acid-resistant coatings. Other suitable enteric coatings include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate and anionic polymers of methacrylic acid and methyl methacrylate. Another suitable enteric coating is a water emulsion of ethylacrylate methylacrylic acid copolymer, or hydroxypropyl methyl cellulose acetate succinate (HPMAS). (See, e.g., U.S. Pat. No. 5,591,433). An enteric coating is designed to resist solution in the stomach and to dissolve in the neutral or alkaline intestinal fluid. In certain embodiments, the bacterial cells are preferably formed into dry powders. Suitable drying methods include a natural drying, a forced-air drying, a spray drying, a freeze drying, and the like. Of those, a spray drying, drum drying or a forced-air drying are preferably used. A protective agent such as skim milk, sodium glutamate, and saccharides may be used in a time of drying. As saccharides, glucose and trehalose may be used.

Auxiliary and adjuvant agents may include, for example, preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents. Prevention of the action of contaminating microorganisms, if desired, can be accomplished using various antibacterial and antifungal agents, such as, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like, may also be included.

Solid dosage forms can be prepared with coatings and shells, such as enteric coatings and others well-known in the art. They can contain pacifying agents and can be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner Examples of embedded compositions that can be used are polymeric substances and waxes. The active compounds also can be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Such dosage forms are prepared, for example, by dissolving, dispersing, etc., the active agent (such as the recombinant bacteria), and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethyl formamide; oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan; or mixtures of these substances, and the like, to thereby form a solution or suspension.

Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art. Reference is made, for example, to Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, Easton, Pa., 1990).

Methods

The methods are directed to treating a parasitic worm or helminth infection in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-microbial agent treated recombinant bacterium that is engineered to express a crystal protein.

Furthmore, the methods are directed to reducing the severity of a parasitic worm or helminth infection comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-microbial agent treated recombinant bacterium that is engineered to express a crystal protein.

Selected Definitions

As used herein, unless the context makes clear otherwise, “treatment,” and similar words such as “treated,” “treating” etc., indicates an approach for obtaining beneficial or desired results, including and preferably clinically desirable results. Treatment can involve optionally either the amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition.

As used herein, unless the context makes clear otherwise, “subject” means a vertebrate, such as a mammal. The mammal can be a feline, a rodent, a canine, a bovine, a equine, a swine, a caprine, an ovine, or a primate. In some embodiments, the subject is a human.

As used herein, unless the context makes clear otherwise, “reducing the likelihood of occurrence,” “prevention,” and similar words such as “prevented,” “preventing” etc., include approaches for preventing, inhibiting, or decreasing the likelihood of the onset or recurrence of a disease or condition, in a manner that exhibits statistical significance, for example, when compared to the results obtained when the indicated method steps are omitted. Similarly, also included are preventing, inhibiting, or decreasing the likelihood of the occurrence or recurrence of the symptoms of a disease or condition, or optionally delaying the onset or recurrence of a disease or condition, or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also include reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition. Methods according to these and related embodiments may be practiced using an effective amount or a therapeutically effective amount of an agent that substantially eradicates, reduces the severity of, or reduces the likelihood of occurrence of a soil-transmitted helminth (STH) infection. As used herein, an “effective amount” or a “therapeutically effective amount” of a composition, agent or substance is that amount sufficient to obtain a desired biological effect, such as beneficial results, including clinical results.

In certain preferred embodiments, the herein described compositions for treating or reducing the severity or likelihood of occurrence of an STH infection are formulated as pharmaceutical compositions, which will preferably be formulated for oral delivery. Pharmaceutical compositions are formulated so as to allow the agent(s) contained therein to be bioavailable upon administration of the composition to a human.

It will be appreciated that the practice of the several embodiments of the present invention will use, unless indicated specifically to the contrary, conventional methods in virology, immunology, microbiology, molecular biology and recombinant DNA techniques that are within the skill of the art, and many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology, 3^(rd) ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other like references.

Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise. Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

EXAMPLES

The following Examples are presented by way of illustration and not limitation.

Example 1 Expression of Cry Proteins in Bacillus

Bacillus thurengiensis, Bacillus cereus (e.g., var. toyoi, var. toyoii), Bacillus toyonensis, Bacillus thuringiensis (e.g., var. HD1), or Bacillus subtilis (e.g., var. PY79, var. natto) is used to express Cry proteins using either sporulation promoters (stationary phase/early sporulation Cry3A, late sporulation Cry5B) or a constitutive promoter (e.g., the mbg promoter). See, e.g., Shao X, et al. “Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor.” MICROB CELL FACT 8: 48 (2009). These constructs are transformed into B. cereus, B. toyonensis, B. thuringiensis, and B. subtilis strains and are tested for expression and bioactivity as described below. In, addition, strong expression promoters (constitutive and inducible) have been made for B. subtilis, and these and other genetic elements described herein are referred to as being “operably linked” when they are present in a polynucleotide construct and situated in a manner that permits them to exert the desired function, such as promotion of specific gene transcription (See, e.g. Phan T T, et al. “Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis.” PROTEIN EXPR PURIF 46: 189-195 (2006). Secreted versions of proteins are made by addition of the signal peptide of the amyQ gene. See id. Thus, similar expression/curative experiments are carried out using Bacillus subtilis as the probiotic strain.

Example 2 Curative Experiment A—Protocol for Infections, Anthelmintic Treatment, and Determination of Treatment Efficacy (Small Intestine Roundworm Parasite)

Six week old female Swiss Webster mice are infected per os with a suspension of 200±10 Heligmosomoides bakeri infective third-stage larvae in 0.1 mL of distilled water. The outbred strain Swiss Webster is used to better “mimic” treating a genetically diverse host (like humans). Each mouse is gavaged on day 15 post-infection (PI) with 0.1 mL of buffer, 0.1 mL of high dose sporulation-defective bacterium such as spo0A- Bacilli (alive or dead) control (transformed with empty vector) or 0.1 mL of high dose bacteria expressing Cry protein (6-10 animals/group). Progression of the infection is determined by fecal egg counts every other day beginning 3 days before treatment. Mice are placed individually in empty plastic cages for 1 h each morning, and the fecal pellets are collected into 50 mL centrifuge tubes. The number of eggs present is counted using the modified McMaster technique. See Hu Y, et al. “Bacillus thuringiensis Cry5B protein is highly efficacious as a single-dose therapy against an intestinal roundworm infection in mice.” PLOS NEGL TROP DIS 4: e614 (2010). At 1, 2, or 3 weeks after treatment, the animals from all three groups are euthanized and the intestinal worm burdens are counted. Using fecal egg counts and intestinal worm burdens, the ability of Cry-expressing sporulation-defective bacterium such as spo0A- Bacilli (alive or dead) to cure small intestinal roundworm infections are ascertained.

Example 3 Curative Experiment B—Trichuris Muris: Whipworm Large Intestine Roundworm Parasite

Twenty-one (21) 6-8 week old female AKR mice are infected per os with 200 infectious-staged T. muris eggs. Thirty (30) days post-infection, the mice are treated per os (7/group) with a single 0.1 mL dose of buffer, 0.1 mL high dose of sporulation-defective bacterium such as spo0A- Bacilli (alive or dead) control (transformed with empty vector), or 0.1 mL of high dose sporulation-defective bacterium such as spo0A- Bacilli (alive or dead) expressing Cry protein. Fecal egg counts are taken three days before treatment and then every other day until necropsy (same protocol to collect eggs as per H. bakeri). The mice are euthanized either 1, 2 or 3 weeks after treatment and worm burdens in the large intestine are determined. Using fecal egg counts and intestinal worm burdens, the ability of Cry-expressing sporulation-defective bacterium such as spo0A- Bacilli (alive or dead) to cure large intestinal roundworm infections are ascertained.

Example 4 Curative Experiment C—Ancylostoma Ceylanicum: Hookworm (Blood Feeding, Small Intestinal Roundworm Parasite)

Twenty one (21) 4-week old Syrian hamsters are infected per os with 150 infectious staged L3 A. ceylanicum hookworm larvae. Fourteen (14) days post-infection, the hamsters are treated per os with a single 0.1 mL dose of buffer, 0.1 mL high dose of sporulation-defective bacterium such as spo0A- Bacilli (alive or dead) control (transformed with empty vector), or 0.1 mL of high dose sporulation-defective bacterium such as spo0A- Bacilli (alive or dead) expressing Cry protein. Body weight, hemoglobin levels, and fecal egg counts (beginning three days before treatment) are monitored every other day until day 21, 28, or 35, at which point the animals are euthanized and worm burdens in the small intestine are determined. Using fecal egg counts, hemoglobin levels, and intestinal worm burdens, the ability of Cry-expressing sporulation-defective bacterium such as spo0A- Bacilli (alive or dead) to cure blood-feeding small intestinal roundworm infections are ascertained.

Studies of Ancylostoma ceylanicum hookworms in Syrian hamsters were carried out as previously described (Hu, Y., et al. PLoS One, 8, e70702, 2013; Hu, Y., et al. Appl Environ Microbiol, 79, 5527-5532, 2013). Briefly, 4-6 week old male hamsters were infected with 150 infectious third staged larvae per os. On day 17 post-infection, an overnight collection of stool is taken and fecal egg counts (FECs) taken the next day. The hamsters are assigned to groups based on FEC so that there is roughly the same level of infection (same average egg per gram of feces or EPG) in all groups. On day 18 post-infection, the hamsters are weighed for dosing purposes and then gavaged with treatments as described in each experiment. On day 21 post-infection, another overnight collection of stool is taken for FECs. On day 22 post-infection, the animals were euthanized. Total hookworms in the small intestine were counted and EPGs calculated.

Studies of Necator americanus hookworms in Syrian hamsters were carried out similarly with the following differences. Following subcutaneous (subq) infection with 150 infectious third staged larvae, hamsters were injected daily with 200 μL of 4 mg/mL dexamethasone to suppress immunological responses that expel the parasites (Fujiwara, R., et al. Parasite Immunol, 28, 285-293, 2006). Treatments were conducted on day 57 post-infection and hookworm burdens/final FECs determined on day 61 post-infection.

For all experiments involving cimetidine, hamsters or mice were pre-gavaged with 200 μL of an 8.75% cimetidine solution 15 minutes prior to therapeutic treatment (Stepek, G., et al. Parasitology, 134, 103-112, 2007).

Example 5 Preventative-Type Experiment A

Swiss Webster mice as above (6-10 each group, three groups) received either 0.1 mL buffer, 0.1 mL high dose empty vector-transformed sporulation-defective bacterium such as spo0A- Bacilli (alive or dead) without Cry protein expression, or 0.1 mL high dose vector-transformed sporulation-defective bacterium such as spo0A- Bacilli (alive or dead) with Cry protein expression. Some (about 2-21) days later, all groups of mice are then challenged with 200 H. bakeri infectious larvae as described above. Two weeks later after infection challenge, fecal egg counts are determined every other day for one to two weeks, after which time the mice are euthanized to determine intestinal roundworm burdens. Fecal egg counts and intestinal roundworm burdens are used to determine if the probiotics protected the mice against a challenge with a small intestine roundworm parasite (i.e., prevented infection).

Example 6 Preventative-Type Experiment B

AKR mice as above (6-10 each group, three groups) receive either 0.1 mL buffer, 0.1 mL high dose empty vector-transformed sporulation-defective bacterium such as spo0A- Bacilli (alive or dead) without Cry protein expression, or 0.1 mL high dose vector-transformed sporulation-defective bacterium such as spo0A- Bacilli (alive or dead) with Cry protein expression. Some (about 2-21) days later, all groups of mice are then challenged with 200 T. muris infectious eggs as above. Thirty (30) days after infection challenge, fecal egg counts are determined every other day for one to two weeks, after which time the mice are euthanized to determine intestinal roundworm burdens. Fecal egg counts and intestinal roundworm burdens are used to determine if the probiotics protected the mice against a challenge with a large intestine roundworm parasite (i.e., prevented infection).

Example 7 Preventative-Type Experiment C

Hamsters as above (6-10 each group, three groups) receive either 0.1 mL buffer, 0.1 mL high dose empty vector-transformed LAB without Cry protein expression, or 0.1 mL high dose vector-transformed sporulation-defective bacterium such as spo0A- Bacilli (alive or dead) with Cry protein expression. Some (about 2-21) days later, all groups of hamsters are then challenged with 150 A. ceylanicum infectious larvae as above. Two weeks after infection challenge, fecal egg counts are determined every other day for one to two weeks, after which time the hamsters are euthanized to determine intestinal roundworm burdens. Fecal egg counts and intestinal roundworm burdens are used to determine if the treatment protected the hamsters against a challenge with a small intestine blood-feeding roundworm parasite (i.e., prevented infection). In addition to experiments with rodents described above, similar experiments could be carried out with other mammals, e.g., felines, canines, bovines, equines, swines, caprines, ovines, and primates.

Example 8 Bacillus Subtilis Strain Engineered for Treatment of STHs

Construction and verification of strains and preparation of lysates. The B. subtilis strain PY79 was transformed with the plasmid vector pHT3101 (PY79-vector) or with a pHT3101-derived cry5B plasmid (PY79-Cry5B) (29). Natural competence was generated in PY79 by use of a standard medium shift protocol (30). To generate spore lysates and spore crystal lysates, PY79 strains were sporulated for 96 h at 37° C., spun down, washed once with prechilled 0.5M NaCl, and washed again with prechilled sterile double-distilled water. Final pellets were stored at −80° C. until use.

Transformants were screened by PCRs using the following primers on all three strains (PY79, PY79-vector, and PY79-Cry5B): Cry5B primer forward 1 (CGTTCAAAATCATCCGTAAATG) (SEQ ID NO: 26) with Cry5B primer reverse 1 (AAATGCATGAACCACTTCCAC) (SEQ ID NO: 27) (predicted product of 586 nucleotides [nt]), Cry5B primer forward 2 (TGGCAACAATTAATGAGT TGTATCCAG) (SEQ ID NO: 28) with Cry5B primer reverse 2 (CTGCCTTGACAAATGG CTACT) (SEQ ID NO: 29) (predicted product of 497 nt), and pHT3101 primer forward (CACCCCAGGCTTTACACTTTA) (SEQ ID NO: 30) with pHT3101 primer reverse (AGG CGATTAAGTTGGGTAACG) (SEQ ID NO: 31) (predicted product of 220 nt with empty vector pHT3101 and 6.5 kb with the cry5B insert). Templates were prepared as follows.

Single colonies of PY79, PY79-vector, and PY79-Cry5B were picked from plates and suspended in 50 μl of sterile double-distilled water. These bacterial solutions were boiled for 3 min and then snap-frozen in liquid nitrogen for 3 min. The procedure was repeated for a total of three cycles of boiling-freezing. Supernatants were collected and used as PCR templates. Cycles were carried out using Taq polymerase under the following conditions: 94° C. for 3 min and then 35 cycles of 94° C. for 30 s, 54° C. for 45 s, and 72° C. for 1 min, followed by 72° C. for 10 min. All amplified products were sequenced to confirm identities. To determine putative transcription factor binding sites, 1.5 kb of the region upstream of the cry5B start codon was entered into the DBTBS database and the P value was set to 0.05. Two putative sigma E binding sites were revealed, 43 and 712 bases upstream of the start codon.

The identity of the strains was further confirmed by analysis of selected proteins. Cell lysates were fractionated by 8% SDS-PAGE, and protein bands were excised from the gels. Proteins were prepared for mass spectrometric sequencing by in-gel digestion with trypsin and then analyzed by high-pressure liquid chromatography (HPLC) in combination with tandem mass spectroscopy (MS/MS) using electrospray ionization as described previously (32). The collected data were analyzed using MASCOT (Matrix Sciences) and Protein Pilot 4.0 (AB Sciex) for peptide identifications.

SEM. In preparation for scanning electron microscopy (SEM) imaging, the samples were drop-cast on a polished Si chip and dried in a vacuum. The samples were then sputter coated with iridium in an Emitech K575X sputter coater. The sputter current was 85 mA, the argon pressure was 2 Pa, and the deposition time was 7 s, resulting in a film thickness of <10 nm. The samples were imaged with an FEI XL30 ESEM FEG instrument, using a 10-kV beam energy and a spot size of 3.

C. elegans bioassays and A. ceylanicum curative experiments. Ancylostoma ceylanicum hookworms were maintained in golden Syrian hamsters (14). All animal experiments were carried out under protocols approved by the UCSD or UMMS Institutional Animal Care and Use Committees (IACUC). All housing and care of laboratory animals used in this study conformed to the Guide for the Care and Use of Laboratory Animals (33) and all requirements and regulations issued by the USDA, including regulations implementing the Animal Welfare Act (P.L. 89-544) as amended (see 18-F23). Caenorhabditis elegans was maintained according to standard procedures (34).

The concentration of Cry5B protein in PY79-Cry5B spore crystal lysates was determined as previously described for Bt Cry5B spore crystal lysates (13). Dose-dependent C. elegans mortality bioassays (three independent trials) were carried out as previously described (13), including use of tetracycline at 30 μg/ml, except that the assays were carried out for 6 days and each well contained ˜25 to 30 animals (with triplicate wells per experiment and three independent experiments). The 50% lethal concentration (LC50) was calculated using PROBIT (35).

For in vivo curative experiments, male hamsters were infected per os with 150 A. ceylanicum infectious larvae. On day 17 postinoculation (p.i.), a fecal sample was collected from each hamster, and the number of eggs was counted using the modified McMaster technique (13). On the basis of these fecal egg counts, the hamsters were segregated to ensure that the groups (control and treatment) had roughly equivalent infection levels. On day 18 p.i., hamsters were weighed individually and given either PY79-Cry5B spore lysate or a spore dose equivalent of PY79-vector spore lysate per os through a blunt-ended gavage needle. Feces were collected on days 1 and 3 post-treatment to determine fecal egg counts (13). The hamsters were sacrificed on day 22 p.i., and intestinal parasite burdens were determined as described previously (14). The one-tailed Mann-Whitney test was performed to compare the two groups for significance in the experiment using a dose of 10 mg/kg of body weight (data were calculated and plotted using Prism 5 [GraphPad Software Inc., La Jolla, Calif.]). Fecal egg counts were compared using one-tailed Student's t test. For the dose-response experiment, results for each treatment group were compared to those for the control group by one-way analysis of variance and Dunnett's method.

Results

Cry5B was well produced in Bacillus subtilis PY79. A recombinant cry5B plasmid engineered for B. thuringiensis (29) was purified from B. thuringiensis and transformed into B. subtilis strain PY79 by standard transformation techniques. This plasmid, based upon the E. coli-B. thuringiensis shuttle vector pHT3101 (36), contained the endogenous Cry5B promoter and 3′-untranslated region driving expression of the wild-type cry5B gene (29). To generate an empty vector control strain, empty vector pHT3101 was also transformed into PY79. The presence of the cry5B gene in the PY79-Cry5B strain and its absence from both the parent PY79 strain and the control strain (PY79-vector) were confirmed by PCR. PCR detection of the plasmid in the PY79-vector strain and its absence from the parent PY79 strain were also confirmed. PY79 was able to maintain both the cry5B plasmid and pHT3101 under standard antibiotic selection with erythromycin, indicating that the origin of replication for B. thuringiensis functioned in B. subtilis, as demonstrated previously (37).

The PY79-Cry5B and PY79-vector strains were sporulated. Robust expression of a protein of the size of Cry5B was detected by PAGE only in the PY79-Cry5B strain. Mass spectroscopy confirmed that the protein was indeed Cry5B. On the basis of quantitation relative to bovine serum albumin (BSA) standards on polyacrylamide gels, Cry5B was expressed at 10 mg/liter culture, which was ˜7.5-fold lower than the Cry5B expression level in B. thuringiensis (75 mg/liter) (29). Two other bands common to both PY79-vector and PY79-Cry5B were identified by mass spectroscopy as the 60-kDa chaperonin protein and an oligopeptide-binding protein from B. subtilis 168, the parent strain of PY79 (38). These assays confirmed that Cry5B was expressed in the PY79-Cry5B strain and that the strain was B. subtilis PY79.

Crystal proteins expressed during sporulation of B. thuringiensis assemble into crystalline inclusions in the mother cell compartment that are often bipyramidal in shape (39). This assembling is also true of Cry5B produced in B. thuringiensis (40). Whereas no crystals were detected by SEM upon sporulation of the PY79-vector strain, many SEM-detectable small crystalline inclusions were present upon sporulation of the PY79-Cry5B strain. Some of these crystals were bipyramidal in shape; others appeared to be truncated versions of such crystals. Thus, Cry5B not only was expressed in PY79 but also assembled into crystalline inclusions.

Cry5B made by PY79 was bioactive. To test whether or not Cry5B made by PY79 was bioactive, dose-dependent mortality assays were set up using the laboratory roundworm C. elegans in a standard 48-well format (13, 41). The Cry5B component of PY79-Cry5B spore crystal lysates was quantitated relative to BSA standards on polyacrylamide gels. Fourth-stage larvae were incubated for 6 days in wells containing PY79-Cry5B spore crystal lysates containing fixed amounts of Cry5B. Antibiotics were included to prevent infection of the roundworms by bacteria (42). Cry5B made by PY79 was found to kill C. elegans, with an LC50 of 4.3 μg/ml (95% confidence interval, 3.6 to 5.0 μg/ml) (FIG. 11). This LC50 was similar to the LC50 of Cry5B purified from B. thuringiensis (7 to 9 μg/ml) (35) under comparable conditions (25° C., 6 days). Conversely, C. elegans exposed to PY79-vector spore lysates (with a spore count equivalent to the highest dose used with PY79-Cry5B) was >99% viable (122/123 worms were alive). Thus, PY79 spore lysates were not lethal to C. elegans, and PY79 was able to produce bioactive Cry5B.

PY79-Cry5B was therapeutic against experimental hookworm infection in hamsters. Nine hamsters were infected with the hookworm parasite A. ceylanicum. At 18 days post-inoculation, five hamsters were treated per os with a single dose of PY9-vector spore lysate, and four were treated with a single dose of PY79-Cry5B spore crystal lysate (equivalent spore counts were used in both treatment groups; the amount of Cry5B was determined relative to BSA standards on protein gels). The single dose of Cry5B used was 10 mg/kg, chosen based on published doses of clinical anthelmintics used in the same model of hookworm disease (Table 3). Feces were collected before and after treatment in order to determine worm loading and changes to parasite egg output. At 22 days post-infection, animals were sacrificed and intestinal worm burdens determined. With a single dose, hookworm burdens were reduced 93% relative to those of the control group (P=0.009) (FIG. 7A). Strong effects could also be seen in the reduction of parasite eggs excreted into feces (91% reduction) (FIG. 7B). To determine if there was an effective dose-response relationship and if significant therapy could be provided at lower doses, another experiment was carried out with three hamsters per group and Cry5B doses of 0.4, 1.4, and 4 mg/kg. Significant clearance of parasites was seen at 1.4 and 4 mg/kg Cry5B in PY79 (69% and 79% reductions, with P values of 0.023 and 0.012, respectively) (FIG. 7C).

The experiments in this Example demonstrated for the first time that Bacillus subtilis can be engineered to provide a significant therapeutic effect against an existing parasitic disease. This pilot study employed PY79, a laboratory strain of B. subtilis that has been used as a model for the delivery of viable bacterial therapies in humans and livestock and that is closely related to a food-grade B. subtilis species. PY79 was made to express and correctly present the BtCry5B protein in a manner that was bioactive against the laboratory roundworm C. elegans. A single 10-mg/kg dose (71 nmol/kg) of Cry5B administered as a Cry5B-PY79 spore crystal lysate reduced A. ceylanicum hookworm burdens in hamsters by 93%, and a dose as small as 1.4 mg/kg was able to provide significant therapy. In previously published data, purified Cry5B delivered at 10 mg/kg reduced hookworm burdens by 65% (14); the data disclosed herein suggest that delivery of Cry5B via PY79 spore crystal lysates was superior to delivery via purified protein.

The expression of Cry5B in B. subtilis employed the endogenous Bt Cry5B promoter and may have been influenced at least partly by two putative sigma E elements upstream of the cry5B start codon. Sigma E is a sporulation-specific promoter that is active in B. subtilis and is also known to be involved in crystal protein production in B. thuringiensis (43, 44). The engineered strain used for the present study included antibiotic resistance genes associated with the cry5B plasmid. Given the genetic tools associated with B. subtilis (45), a Cry5B-expressing B. subtilis therapeutic product for humans is contemplated that includes the cry5B gene integrated into the genome and that lacks any antibiotic resistance genes

The 93% elimination (P=0.0.009) of A. ceylanicum hookworm parasites from hamsters by use of a single 10-mg/kg (71 nmol/kg) dose compared favorably to the results of anthelmintics used clinically (Table 3). For example, a 10-mg/kg (49 μmol/kg) dose of levamisole resulted in a 60% reduction of A. ceylanicum burdens in hamsters, a 10-mg/kg (17 μmol/kg) dose of pyrantel resulted in an 87% reduction in A. ceylanicum burdens, a 10-mg/kg (22 μmol/kg) dose of tribendimidine resulted in a 75% reduction of A. ceylanicum burdens, and a 1.25-mg/kg (4.7 μmol/kg) dose of albendazole resulted in an 88% reduction of A. ceylanicum burdens (46, 47). In addition to high efficacy, Cry5B had a different mechanism of action from that of chemical anthelmintics; Cry5B has been shown to be a pore-forming protein that binds to invertebrate-specific glycolipids and attacks the plasma membrane of the nematode intestine (34, 35, 48-50).

As described herein PY79-Cry5B was comparable to many current drugs in its efficacy on a mg/kg basis, and on a molar level, it appeared to be superior (e.g., the molar dose of Cry5B used in the present experiments was 66 times lower than the molar dose of albendazole mentioned above). The present results validated the B. subtilis-Cry5B approach.

Also contemplated are increasing B. subtilis-Cry5B specific activity, e.g., by Cry5B point mutations that increase roundworm-killing activity (51) and by optimization of fermentation conditions that can also increase crystal protein specific activity (52). Given that Bacillus bacteria can be produced and stored cheaply and in large quantities (53), the present results demonstrated the feasibility of Cry5B delivery by food-grade B. subtilis for the treatment of STH diseases.

TABLE 3 Comparison of efficacies of PY79-Cry5B and clinically used anthelmintics against A. ceylanicum infections in hamsters Dose % Parasite Treatment^(a) (_mol/kg) reduction P value Reference Levamisole 49 60 0.057 47 Pyrantel 17 87 0.057 47 Tribendimidine 22 75 >0.05? 46 Albendazole 4.7 88 <0.001 47 (1.25 mg/kg) Cry5B 0.071 93 0.009 This Example ^(a)Treatments were administered at 10 mg/kg unless otherwise stated.

Example 9 Bioactivity of Compositions Comprising Cry5B and Bacteria

This example describes additional data that were obtained using the above-described bioassays for anthelmintic activity.

FIG. 8 shows results from an in vivo bioassay of intestinal worm burdens in golden Syrian hamsters infected with Ancylostoma ceylanicum hookworms as described by Hu et al. (2012 PLoS Negl. Trop. Dis. 6:e1900. doi:10.137/journal.pntd.0001900). The four groups (in black, n=7 per group) shown are the intestinal worm burdens from the groups of infected hamsters treated with purified full-length B. thuringiensis Cry5B protein (prepared according to Griffitts et al., 2001 Science 293:860; for sequence see FIG. 2) at a single dose of 1 mg (solid circles, 10 mg/kg), 3 mg (solid upright triangles, 30 mg/kg), or 10 mg (solid inverted triangles, 100 mg/kg) (715 nmoles/kg), or with placebo (solid squares, ddH₂O), respectively. The treatments were conducted on day 16 P.I. and intestinal worm burdens assessed on day 21 P.I. The worm burdens in each hamster are indicated with a separate symbol. Long horizontal bars represent mean worm burdens; smaller bars indicate SEM (standard error of the mean).

FIG. 9 shows dose-response results for indicated dosages of unfractionated Cry5B-containing spore-crystal lysates (SCL) in the in vivo bioassay of intestinal worm burdens in golden Syrian hamsters infected with Ancylostoma ceylanicum hookworms. The assay was performed according to Hu et al. (2012 PLoS Negl. Trop. Dis. 6:e1900. doi:10.137/journal.pntd.0001900) except instead of purified Cry5B protein the animals received the indicated dosages, via gavage, of Cry5B spore-crystal lysates obtained from cultured Bacillus thuringiensis cells that were transformed with a low copy plasmid that expressed B. thuringiensis Cry5B and then grown to sporulation phase. The amounts of Cry5B gavaged were determined by taking known volumes of spore crystal lysates, resolving full length Cry5B protein by SDS PAGE, and quantitating the amount of protein in the Cry5B band relative to known amounts of bovine serum albumin (BSA) standards on the gel.

FIG. 10 shows results from the in vivo bioassay of intestinal worm burdens in golden Syrian hamsters infected with Ancylostoma ceylanicum hookworms (Hu et al., 2012 PLoS Negl. Trop. Dis. 6:e1900. doi:10.137/journal.pntd.0001900) following treatment with two different dosages of Cry5B spore-crystal lysates obtained from cultured Bacillus thuringiensis natto cells that were transformed with a low copy plasmid that expressed B. thuringiensis Cry5B and then grown to sporulation phase. B. subtilis natto was transformed with the same Cry5B expressing plasmid described in Example 14 (Hu et al. Appl. Environ. Microbiol. 2013, 79(18):5527). Because B. subtilis natto is not naturally competent, B. subtilis natto cells were made competent by artificially introducing the ComK competency plasmid into the B. subtilis natto strain via protoplast transformation (Ashikaga et al., J Bacteriol. 2000; 182(9):2411-5; Romero, D., et al J Microbiol Meth. 2006; 66(3):556-9). The resultant strain was able to take up any DNA and the ComK plasmid, being unstable, was readily lost by growing under non-selective pressure).

FIG. 11 shows data obtained in vitro using the C. elegans mortality assay described in FIG. 6 to evaluate the effects on C. elegans of purified Cry5B protein (prepared according to Griffitts et al., 2001 Science 293:860; for sequence see FIG. 2) when combined in a mixture either with sporulated B. thuringiensis HD1 or with sporulated B. subtilis PY79. For each data point, the number of spores (HD1 or PY79) was held constant and the quantity of Cry5B was titrated (x-axis).

FIG. 12 shows results from the in vivo bioassay of intestinal worm burdens in golden Syrian hamsters infected with Ancylostoma ceylanicum hookworms (Hu et al., 2012 PLoS Negl. Trop. Dis. 6:e1900. doi:10.137/journal.pntd.0001900) following treatment (5 mg/kg) by gavage on day 18 P.I. with either B. thuringiensis strain HD1 spore lysates transformed with empty vector (“control”, spore lysates from the acrystaliferous mutant B. thuringiensis strain HD1, which does not produce any Cry proteins) or spore crystal lysates from B. thuringiensis strain HD1 that has been engineered to express Cry14A (for sequence see FIG. 4) using a plasmid encoding Cry14A under the control of the operably linked Cry3A promoter. Hookworm burdens were assessed on day 20 post-infection (P.I.).

Example 10 Gene Replacement and Generation of a Bacillus Subtilis Auxotroph

Cry5B gene was integrated into the B. subtilis genome by a strategy that simultaneously deleted the chromosomal thyA gene, which encodes thymidylate synthetase. A cry5B cassette, flanked by the upstream and downstream regions of B. subtilis thyA, was assembled in vitro by standard PCR techniques. B. subtilis natto was transformed with this construct in a single step. Transformants simultaneously acquired two properties: auxotrophy for thymine nucleotides and the production of Cry5B protein. Because thymine auxotrophs in B. subtilis are known to be naturally resistant to trimethoprim and other antifolate compounds, selection for growth in the presence of trimethoprim plus thymine selected for the desired integration event without the introduction of an antibiotic resistance marker. The construct contained no foreign DNA at all except for the cry5B gene itself. The auxotroph permitted easy replication under laboratory conditions but the strain was environmentally dead and unable to replicate in the wild (e.g., following defecation by a human).

Example 11 Generation of a Cry5B-Expressing B. Thuringiensis Cell Effective in Treating Parasitic Worm/Helminth Infection

Cry5B crystals are formed and trapped into a sporulation-deficient spo0A- B. thuringiensis cell when Cry5B is expressed under the control of a stationary phase promoter like Cry3A; such a bacterium was made using standard molecular biology techniques and used the construct depicted in the center panel of FIG. 13. The expression of the protein was good (FIG. 18C). The cells were treated with terpenes (carvacrol) for 15 or 60 minutes; the level and integrity of Cry5B crystals does not change with this treatment (FIG. 18C). Carvacrol teatment killed the recombinant cells (FIG. 18B). Based on C. elegans assays and in vivo hookworm curative studies in hamsters, the carvacrol treated and untreated cells were bioactive against nematodes (FIGS. 18-22).

Example 12 Production of Cry5B Spore Crystal Lysate and Purified Protein

Cry5B spore-crystal lysates (SCL) were produced by sporulation in PGSM medium as previously described (Cappello, M., et al. Proc Nat Acad Sci. 103, 15154-15159, 2006; Marroquin, L., et al. Genetics. 155, 1693-1699, 2000). The Cry5B promoter and gene were cloned into vector pHT3101 (Lereclus, D., et al. FEMS Microbiol. Lett. 60, 211-217, 1989) and transformed into Crystal protein minus (Cry−) Bacillus thuringiensis (Bt) subsp. kurstaki strain 4D8 (from the Bacillus Genetics Stock Center) using standard protocols. When required, Cry5B protein was purified from SCL using published protocols (Griffitts, J., et al. Science. 293, 860-864, 2001).

Example 13 Production of BaCC (Bacillus with Cytosolic Crystal) and IBaCC (Inactivated Bacillus with Cytosolic Crystal)

BaCC

For production of Cry5B in sporulation defective cells, Bt strain 407 lacking the master spo0A regulator of sporulation (Lereclus, D., et al. Nat. Biotechnol. 13, 67-71, 1995) was transformed using electroporation with Cry5B cloned into the pHT3101 vector under control of the Cry3A promoter (Agaisse and Lereclus, J. Bacteriol. 176, 4734-4741, 1994; Agaisse and Lereclus, Mol. Microbiol. 13, 97-107, 1994) to create the Bt-spo0A- Cry5B strain. This composition is further referred to as Cry5B-BaCC (Bacillus with Cytosolic Crystal).

IBaCC

B. thuringiensis 407 strains were propagated aerobically in 200 mL volume in 2 liter baffled flasks with shaking at 30° C. in three-fold concentrated Luria-Bertani broth (LB) supplemented with 10 μg/mL erythromycin and 200 μg/mL kanamycin for 48 hours. The B. thuringiensis cells were spun down at 4500 rpm for one hour at 4° C. and resuspended to ¼ of the original cell culture volume with prechilled sterile double-distilled water, and then were treated with 1 mg/mL carvacrol for 15 min with shaking at 4° C. The carvacrol treated cells were spun down and washed three times with prechilled sterile double-distilled water. Final pellets were concentrated 40 times and were stored at −80° C. until use. A diagram showing the IBaCC process is shown in FIG. 18A.

Freeze Drying

Samples were frozen at −80 C and then put into a FreeZone 1 Liter Benchtop Freeze Dry System (Labconco catalog number 7740020). The condenser was set to −60° C. and the vacuum at 22 mTor. The samples were freeze-dried overnight.

Spray-Drying

For spray-dried IBaCC, a 400 mL IBaCC sample at 10% solids w/v is spray dried using a Yamato Pulvis GB22 (or any other spray drying system) through a 100 micron atomizer nozzle at 5 mLs/min with atomizing air set at 1 Kgf/m², drying air set at 0.21 m³/min, inlet/outlet temperatures set at 98° C./59° C., respectively.

Example 14 Terpene Mediated Inactivation of BaCC

Minimum Inhibitory Concentration

Three terpenes were tested against BaCC including: carvacrol, geraniol and farnesol, by using the broth microdilution method as described in Agaisse and Lereclus, J. Bacteriol, 176, 4734-4741, 1994 and Agaisse and Lereclus, Mol. Microbiol. 13, 97-107, 1994 with some modifications. Briefly, BaCC from a single colony was grown in 5 mL of LB medium supplemented with 10 μg/mL erythromycin+200 μg/mL kanamycin overnight (O.D. (600) of 4.8, 2.3×10⁷ CFU/mL). Terpene solutions were prepared at 1 mg/mL in LB. One hundred μL of two-fold serial dilutions of each terpene in LB were prepared in 96-well microtiter plates over the range of 1000 to 3.9 μg/mL in triplicate. LB without terpenes was used as a negative control. BaCC culture was diluted 100 times, and 100 μL of BaCC culture was added to the wells. Absorbance at 600 nm was recorded before and after the incubation to monitor bacterial growth. The MICs were determined after incubation for 24 h at 30° C. without shaking. The minimum terpene concentration resulted in no absorbance increase overnight reported as minimum inhibitory concentration (MIC).

Time to Kill

In order to determine which terpene kills BaCC in the shortest amount of time, we incubated 5 mL of BaCC culture with three terpenes at 1 mg/mL at 4° C. for 1, 2, 3 and 5 hours. No terpene control and no bacteria controls were carried out simultaneously. After incubation with terpenes, samples were centrifuged and the pellets were washed with 5 mL of 1M NaCl once and 5 mL of sterile distilled water twice in order to remove the terpenes. The pellets were re-suspended in the original volume and plated on LB agar with kanamycin and erythromycin. Plates were incubated overnight at 30° C.; the next day, the number of colonies on the plates were enumerated and colony forming units (CFU)/mL calculated.

To assess terpene killing at shorter time points, BaCC culture was grown in 3×LB for 48 h as described above and 60 ml of cultures were aliquoted in sterile tubes. Carvacrol and geraniol were added to the final concentration of 1 mg/mL, and the tubes were placed on a rotator at 4° C. for 15 minutes. The negative control was prepared using sterile water instead of terpenes. At the end of the incubation period, the tubes were centrifuged at 2000×g for 10 minutes and pellets washed with 60 ml of 1 M NaCl once and washed with 60 mL of sterile distilled water twice. Pellets were re-suspended in 1.2 mL of sterile distilled water to concentrate the culture 50 times. Concentrated terpene treated and control samples were plated on LB agar plates containing kanamycin and erythromycin and incubated overnight at 30° C. The number of colonies were counted the next day and CFU/mL calculated for each time point.

All 3 terpenes had antimicrobial activity but carvacrol was the most efficient, killing the quickest and at the lowest concentration as depicted in Table 4 below.

TABLE 4 Terpene treatments of Bt CFU/ml MIC (1 mg/mL terpene treatment for time indicated) ug/mL Samples 0 15 min 1 h 3 h 5 h (100) No Bacteria 0 0 0 0 control Bt control 2.31 × 1.50 × 1.00 × 1.60 × (no terpene) 10⁷ 10⁷ 10⁷ 10⁷ Carvacrol 2.31 × 0 0 0 125 10⁷ Farnesol 2.31 × 1.00 × 1.3 × 0 500 10⁷ 10⁷ 10⁵ Geraniol 2.31 × 0 0 0 500 10⁷ Bt control 9.27 × (no terpene; 10⁸ 50× conc.) Carvacrol  0 (50× conc.) Geraniol 100 (50× conc.) Terpene Washing Protocol

To reduce carvacrol residue in the final product, we tested different strategies. In order to reduce the carvacrol residue in the final IBaCC product, first the total amount of carvacrol was reduced by concentrating the cultures before carvacrol treatment. The cultures were centrifuged, and the pellets were re-suspended in sterile distilled water in half or one fourth of the original culture volume. Then, carvacrol was added at 1 mg/mL concentration. Second, carvacrol was removed from the culture by increasing the solubility of carvacrol in the water phase. Carvacrol is a lipophilic compound with a log P octanol/water of 3.64; therefore, it is held in the cell pellet more than the aqueous phase. As such, 5% and 15% of ethanol water was used in the washing steps following the IBaCC incubation with carvacrol. Another strategy employed was to increase the volume of distilled water during the washing steps to remove more carvacrol per wash step (for instance, only 25 mg carvacrol can be solubilized with 20 mL of water while 50 mg carvacrol can be solubilized with 40 mL of water.) Lastly, since the temperature affects carvacrol solubility, the incubation and washing steps were carried out at room temperature instead of 4° C. The positive control sample was treated with 1 mg/mL carvacrol without concentrating the culture prior to carvacrol treatment, incubated at 4° C., washed with 1M NaCl once and distilled water twice using 1:1 culture to wash volume ratio. No terpene control sample received water instead of carvacrol, but the incubation and washing steps were carried out in the same manner as the positive control. BaCC culture was grown in 3×LB for 48 hours, and 40 mL of BaCC culture was used for each condition. After washes, the culture was concentrated 20 times. Samples were plated for CFU/mL analysis as described earlier, and carvacrol residue was assessed by HPLC as follows.

IBaCC concentrates and no carvacrol control BaCC were diluted 1:10 in methanol and incubated one hour on a rotator in order to extract carvacrol. Then, the tubes were centrifuged and the supernatants were collected for HPLC analysis. Carvacrol standards ranging from 0-1000 μg/mL were prepared in 90% methanol-water. IBaCC, control and carvacrol standards were analyzed using RP-HPLC. The HPLC instrument consisting of a Beckman System Gold consisting of a 126 NMP Solvent Module, a 168 NM photodiode array detector and a 508 autosampler with Beckman System Gold 32 Karat 7.0 Data System software. Analysis was carried out using the stationary phase of a C18 column (μBondapak, Waters, 3.9 mm×300 mm, 10 μm) and the mobile phase of acetonitrile:water (70:30) in isocratic mode at 1 mL/min; the injection volume of 10 μL and the detection wavelength were set at 210 nm. The carvacrol concentration of each sample was determined from the corresponding peak area of the carvacrol standard curve.

TABLE 5 Effects of varying carvacrol wash protocols Carvacrol Number Number residue in the of alive of alive 20X bacteria in bacteria in concentrate 1 ml 1 ml 20X after washes Condition culture concentrate (ug/ml) Concentrated 2X = 20 ml 0 0 78.66 before adding carvacrol Concentrated 4X = 10 ml 0 0 11.29 before adding carvacrol Washes were done with 0 0 91.03 5% ethanol in water Washes were done with 8 160 57.75 15% ethanol in water Wash volume increased 7 140 27.78 2X (e.g., 40 ml wash for 20 ml culture) Incubated and washed at 0 0 16.48 room temperature Control: 4C, no 0 0 52.10 concentration, 1:1 culture to wash volume no carvacrol control 1.80 × 10⁸ 3.60 × 10⁹ 0

Example 15 Irradiation Mediated Inactivation of Bt

Gamma irradiation was carried out with a cobalt using freeze-dried Cry5B. A dose of 15 kGy was tested on SCLs of three different Bt strains expressing Cry5B or vector control. FIG. 16 A-B shows that this dose of gamma irradiation did not effectively completely kill the Bt strains nor did the strains retain full Cry5B bioactivity (no detectable bioactivity was seen). Further testing at doses ranging from 5-60 kGy did not significantly improve the results.

Example 16 Cry5B Spore Crystal Lysate Treatment In Vivo

The normal life cycle of Bacillus thuringiensis (Bt) is such that when it runs out of nutrients, it enters the sporulation cycle during which time it produces insecticidal/nematicidal crystal (Cry) proteins packaged in a crystal. When the mother cell lyses upon completion of sporulation, the crystal and spore are released, along with bacterial lysate, giving rise to spore-crystal lysate (SCL), shown in FIG. 15A. A mother cell 180, enters the sporulation cycle, and produces a crystal protein 182 and a spore 184. Later during sporulation, mother cell 180 breaks down, releasing spore 184, protein crystal 182, and lysate 186.

It was previously discovered that Bacillus thuringiensis (Bt) spore crystal lysates (SCLs) are highly effective against Heligmosomoides polygyrus bakeri infections in vivo (Hu, Y., et al. Proc Natl Acad Sci. 107, 5955-5960, 2010). Furthermore, a single 40 mg/kg dose cures Ancylostoma ceylanicum hookworm infections in hamsters, shown in FIGS. 15B and 15C. This activity is clearly superior to that of purified Cry5B protein (Hu, Y., et al. PLoS Negl. Trop. Dis. 6, e1900, 2012). One advantage of Bt over other bacteria that can be engineered to make Cry protein [e.g., Bacillus subtilis, (Hu, Y., et al. Appl. Environ. Microbiol. 79, 5527-5532, 2013)] is that crystal yields are much higher in Bt and batch-to-batch reproducibly is superior. This may be due to the fact that Bt is the natural host for Cry protein production and is naturally evolved to produce these proteins at high levels.

Example 17 BaCC Treatment In Vivo

Although Bacillus spores are difficult to kill, vegetative Bacilli are relatively easy to kill. Since Bt Cry proteins can be produced in non-sporulating Bt under a stationary-phase promoter (Agaisse and Lereclus, J. Bacteriol. 176, 4734-4741, 1994; Agaisse and Lereclus, Mol. Microbiol. 13, 97-107, 1994), such Bt cells might be much easier to kill in a manner that would retain Cry5B bioactivity. Cry5B was cloned under the control of the Cry3A stationary-phase promoter and transformed the construct into sporulation-defective (spo0A-) Bt cells (Agaisse and Lereclus, J. Bacteriol. 176, 4734-4741, 1994; Agaisse and Lereclus, Mol. Microbiol. 13, 97-107, 1994). These cells, which do not sporulate, produce Cry5B crystals trapped in the cytosol. Cry5B produced in these cells is intoxicating to the laboratory nematode Caenorhabditis elegans, shown in FIG. 17A and it is bioactive against A. ceylanicum hookworm infections in vivo, shown in FIG. 17B-C. Such cells are termed BaCC for Bacillus with Cytosolic Crystal.

Example 18 IBaCC Treatment In Vivo

Carvacrol treatment of BaCC (spo0A- cells expressing Cry5B) cells resulted in complete killing of the Bacilli, shown in FIG. 18B while Cry5B protein as viewed on polyacrylamide gels looked normal, shown in FIG. 18C. Killed BaCC was still active against C. elegans and against A. ceylanicum hookworm infections in hamsters, shown in FIG. 18D-E. Killed BaCC was equally potent as live BaCC against hookworm infections in vivo, shown in FIGS. 18F-G. The dead BaCC containing biologically active Cry5B crystals are termed IBaCC for Inactivated Bacillus with Cytosolic Crystal.

Example 19 Neutralization of Stomach Acid Prior to BaCC or IBaCC Treatment In Vivo

Since it was not possible to successfully encapsulate Cry5B to protect it from stomach acid for rodent delivery, neutralization of stomach acid was tested to improve Cry5B efficacy. Hamsters were infected with A. ceylanicum hookworms and either neutralized stomach acid or not by pre-gavaging with sodium bicarbonate (NaHCO₃) and cimetidine, and then treated the infected animals with Cry5B purified protein. Pre-neutralization of stomach acid improved Cry5B efficacy FIG. 19A-B. Similarly it was found that pre-neutralization of stomach acid improved the efficacy of Cry5B delivered as Bt spore-crystal lysates (SCL), shown in FIGS. 19C-D and Cry5B delivered as IBaCC, shown in FIG. 19E-F.

Example 20 Freeze-Dried or Spray-Dried IBaCC Treatment In Vivo

For future product development Cry5B would have to be stored/delivered in a dry powder form. To test that Cry5B can be dried into a power and still retain bioactivity, Cry5B IBACC powder was prepared by freeze-drying. This powder, when resuspended in water, retains full bioactivity against hookworm infections in vivo FIG. 21A-B. The near complete clearance seen with 6 mg/kg freeze-dried Cry5B IBaCC is equivalent in mass to the dose of albendazole (5 mg/kg) required to clear the same infection in hamsters. Given that Cry5B is ˜500× larger molecular weight than albendazole, then these data demonstrate that Cry5B IBaCC is a highly potent nematocide on a molecule by molecule basis.

A 400 mL IBaCC sample at 10% solids w/v was also spray dried using a Yamato Pulvis GB22 through a 100 micron atomizer nozzle at 5 mLs/min with atomizing air set at 1 Kgf/m², drying air set at 0.21 m³/min, inlet/outlet temperatures set at 98° C./59° C., respectively. Yield was 30.5 g of a fine, light brown powder.

Example 21 Effects of the Immune System on Cry5B Treatment

Necator americanus is by far the dominant hookworm species infecting humans. The sensitivity of N. americanus to anthelmintics is critically important for their acceptability for STH treatment. For example, the lack of efficacy of ivermectin against Necator (but not Ancylostoma) hookworms is an important reason why ivermectin is not one of the WHO approved anthelmintics for STH treatment. To test if Cry5B is active against Necator hookworms, we infected hamsters with a strain of Necator americanus recently taken from human stool specimens and adapted to immunosuppressed hamsters. To maintain the hookworms in the hamsters, hamsters were immunosuppressed daily with dexamethasone. Despite the immunosuppression, we found that both Cry5B SCL, shown in FIGS. 22A-B, and Cry5B IBaCC, shown in FIG. 22C-D, were effective at single doses against N. americanus hookworms. Thus, Cry5B is effective against both major hookworm genuses represented by three hookworm species in mammalian hosts.

The fact that Cry5B is effective against N. americanus in immunosuppressed hamsters demonstrates that Cry5B does not require the immune system to repulse the parasites. Such a result is not surprising given that Cry5B directly intoxicates STH parasites in vitro. This is not true of all anthelmintics, e.g., DEC and in some cases ivermectin, for which it appears that involvement of the immune system is important for their mechanism of action. To confirm that the immune system is not essential for Cry5B action, STAT6-/-mice were infected with Heligmosomoides polygyrus STH parasites. STAT6 is required for the TH2 response required for normal expulsion of STH parasites. FIG. 23A-B shows that Cry5B is effective against H. polygyrus even in immunosuppressed mice lacking STAT6-/-. Thus, based on the effectiveness of Cry5B in these two different immunosuppressed systems, we conclude that Cry5B acts directly on the parasites and does not require a fully intact immune system for efficacy.

Example 22 Effects of Repeated Doses of Cry5B Treatment on Animals

Although Cry5B does not require the immune system to act, the immune system might still interact with Cry5B and neutralize its activity. Whether or not repeated exposure to Cry5B might result in an adverse immunological response in the animal that would reduce its efficacy was tested. To assess this, hamsters were pre-exposed to two doses of Cry5B prior to infection with A. ceylanicum hookworms. A control group did not receive the pre-exposure to Cry5B but did receive the hookworm infection. Both groups were then treated therapeutically with Cry5B either as pure protein, shown in FIG. 24A-B or SCL, shown in FIG. 24C. In both cases, the efficacy of Cry5B was not altered by pre-exposure to Cry5B. These data demonstrate that Cry5B will be effective over repeated doses.

Example 23 Effects of Cry5B Treatment on Hookworm Infection in Dogs

Two beagles were inoculated per os with 1000 Ancylostoma caninum larvae. Forty one days post-infection, stool was collected for determination of fecal egg loads and the dog intestines were imaged using capsule endoscopy to count hookworms (Lee, A., et al. Int. J. Parasitol. 41, 1377-1383, 2011). One dose of Cry5B SCL (˜40 mg/kg) was given by gavage on each of day 42 and 44 post-infection. On day 49 post-infection a final fecal egg count was taken and capsule endoscopy repeated for final hookworm counts. Table 6 below shows that Cry5B treatment was highly effective against A. caninum in dogs.

TABLE 6 A. caninum fecal egg count and hookworm burden reduction in dogs (n = 2) following treatment with 40 mg/kg Cry5B Fecal Egg Count (EPG of Feces) Hookworm Burden Pre- Pre- Treatment Post-Treatment Treatment Post-Treatment Dog 1 15750 1590 84 4 Dog 2 9412 573 56 3 Average 12581 1081.5 70 3.5 % Reduction — 91.4% — 95%

Example 24 Anti-Nematode Efficacy of BaCC on C. Elegans

Synchronous fourth larval staged hermaphrodites were placed in wells with OP50 Escherichia coli as a food source plus appropriate test article (e.g., BaCC or IBaCC) and tetracycline to prevent infection by any of the bacteria. To assess the effects of Cry5B-BaCC on the laboratory nematode, C. elegans were incubated with 100 μg/mL Cry5B-BaCC for 24 hours at 25° C. Cry5B-BaCC was intoxicating to C. elegans as depicted in FIGS. 17E-F.

Example 25

Bioactivity of carvacrol-treated Crystal-E. coli on C. elegans. Escherichia coli cells expressing nematicidal Crystal protein or empty vector were treated for 15 minutes with 1 mg/mL carvacrol. The cells were washed in sterile water 3 times. CFUs were determined for untreated and carvacrol-treated cells by serial dilution plating on LB plates. In addition, C. elegans nematode L4 hermaphrodites were fed overnight 1.8×10⁷ cells/mL of E. coli untreated or carvacrol-treated, Crystal expressing and empty vector (4 conditions) at 25° C. for 16 hr. As shown in FIG. 25B, empty vector untreated/treated had no impact on the health of C. elegans but both carvacrol-treated/untreated Crystal-expressing E. coli intoxicated C. elegans completely. In addition, as shown in FIG. 25A treatment of the E. coli with carvacrol effectively killed the E. coli.

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All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

While particular steps, elements, embodiments and applications of the present invention have been shown and described herein for purposes of illustration, it will be understood, of course, that the invention is not limited thereto since modifications may be made by persons skilled in the art, particularly in light of the foregoing teachings, without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure. 

The invention claimed is:
 1. An orally-available pharmaceutical composition comprising a killed, non-sporulating bacterium that expresses a nematicidal protein, wherein the bacterium has a genetic mutation comprising a deletion or inactivation of one or more genes selected from the group consisting of kinA, kinB, spo0A, spo0B, spo0E, spo0F, spo0J, spo0M, spoIIB, spoIID, spoIIE, spoIIF, spoIIG, spoIIL, spoIIM, spoIIIA, spoIIIB, spoIIIE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK that results in a defect in sporulation, wherein the nematicidal protein is a cytoplasmic crystal (Cry) protein selected from the group consisting of Cry5B, Cry5C, Cry5D, Cry6A, Cry13A, Cry14A, Cry21A, and Cry 21B, and wherein the nematicidal protein is trapped in the cytosol of the bacterium.
 2. The pharmaceutical composition of claim 1, wherein a nematicidal gene encoding the nematicidal protein is under control of a non-sporulation-specific promoter.
 3. The pharmaceutical composition of claim 1, wherein the bacterium is a Gram-positive bacterium.
 4. The pharmaceutical composition of claim 1, wherein the composition is encapsulated by a pharmaceutical grade capsule in a dry powdered form.
 5. The pharmaceutical composition of claim 1, wherein the bacterium is a species of Bacillus.
 6. The pharmaceutical composition of claim 1, wherein the bacterium is Bacillus thuringiensis (Bt).
 7. A method for producing the pharmaceutical composition of claim 1, the method comprising: (a) exposing a non-sporulating bacterium to an antimicrobial agent, thereby killing or inactivating the bacterium, wherein the bacterium is genetically engineered to express a nematicidal protein and wherein the bacterium has a genetic mutation that prevents sporulation such that the nematicidal protein is trapped in the cytosol of the bacterium; and (b) formulating the killed or inactivated bacterium in an orally-available dosage form.
 8. The method of claim 7, wherein formulating comprises one or more of: (a) lyophilizing or spray drying the bacterium; or (b) encapsulating the bacterium in a pharmaceutical-grade capsule.
 9. The method of claim 7, wherein the antimicrobial agent is selected from the group consisting of: an antimicrobial compound and gamma irradiation.
 10. The method of claim 9, wherein the antimicrobial compound is: (a) a food-grade antibiotic; (b) a beta-lactam antibiotic; or (c) a terpene, iodine or formaldehyde.
 11. The method of claim 10, wherein the terpene is selected from the group consisting of thymol, eugenol, geraniol, carvacrol, and citral, and combinations thereof.
 12. A method of treating a parasitic worm infection in a subject comprising administering to the subject the pharmaceutical composition of claim
 1. 